Cold-adapted equine influenza viruses

ABSTRACT

The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions. Such methods include using a therapeutic composition as a vaccine to generate a protective immune response in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recently infected with a virulent virus, or is likely to be subsequently exposed to virulent virus in a few days whereby the therapeutic composition interferes with the growth of the virulent virus, even in the absence of immunity. The present invention also provides methods to produce cold-adapted equine influenza viruses, and reassortant influenza A viruses having at least one genome segment of an equine influenza virus generated by cold-adaption.

FIELD OF THE INVENTION

The present invention relates to experimentally-generated cold-adaptedequine influenza viruses, and particularly to cold-adapted equineinfluenza viruses having additional phenotypes, such as attenuation,dominant interference, or temperature sensitivity. The invention alsoincludes reassortant influenza A viruses which contain at least onegenome segment from such an equine influenza virus, such that thereassortant virus includes certain phenotypes of the donor equineinfluenza virus. The invention further includes genetically-engineeredequine influenza viruses, produced through reverse genetics, whichcomprise certain identifying phenotypes of a cold-adapted equineinfluenza virus of the present invention. The present invention alsorelates to the use of these viruses in therapeutic compositions toprotect animals from diseases caused by influenza viruses.

BACKGROUND OF THE INVENTION

Equine influenza virus has been recognized as a major respiratorypathogen in horses since about 1956. Disease symptoms caused by equineinfluenza virus can be severe, and are often followed by secondarybacterial infections. Two subtypes of equine influenza virus arerecognized, namely subtype-1, the prototype being A/Equine/Prague/1/56(H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8).Presently, the predominant virus subtype is subtype-2, which has furtherdiverged among Eurasian and North American isolates in recent years.

The currently licensed vaccine for equine influenza is an inactivated(killed) virus vaccine. This vaccine provides minimal, if any,protection for horses, and can produce undesirable side effects, forexample, inflammatory reactions at the site of injection. See, e.g.,Mumford, 1987, Equine Infectious Disease IV, 207-217, and Mumford, etal., 1993, Vaccine 11, 1172-1174. Furthermore, current modalities cannotbe used in young foals, because they cannot overcome maternal immunity,and can induce tolerance in a younger animal. Based on the severity ofdisease, there remains a need for safe, effective therapeuticcompositions to protect horses against equine influenza disease.

Production of therapeutic compositions comprising cold-adapted humaninfluenza viruses is described, for example, in Maassab, et al., 1960,Nature 7, 612-614, and Maassab, et al., 1969, J. Immunol. 102, 728-732.Furthermore, these researchers noted that cold-adapted human influenzaviruses, i.e., viruses that have been adapted to grow at lower thannormal temperatures, tend to have a phenotype wherein the virus istemperature sensitive; that is, the virus does not grow well at certainhigher, non-permissive temperatures at which the wild-type virus willgrow and replicate. Various cold-adapted human influenza A viruses,produced by reassortment with existing cold-adapted human influenza Aviruses, have been shown to elicit good immune responses in vaccinatedindividuals, and certain live attenuated cold-adapted reassortant humaninfluenza A viruses have proven to protect humans against challenge withwild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol.23, 73-76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep.22, 1992, the inventors of the present invention further demonstratedthat certain reassortant cold-adapted human influenza A viruses alsopossess a dominant interference phenotype, i.e., they inhibit the growthof their corresponding parental wild-type strain, as well asheterologous influenza A viruses. U.S. Pat. No. 4,683,137, by Coggins etal., issued Jul. 28, 1987, and U.S. Pat. No. 4,693,893, by Campbell,issued Sep. 15, 1987, disclose attenuated therapeutic compositionsproduced by reassortment of wild-type equine influenza viruses withattenuated, cold-adapted human influenza A viruses. Although thesetherapeutic compositions appear to be generally safe and effective inhorses, they pose a significant danger of introducing into theenvironment a virus containing both human and equine influenza genes.

SUMMARY OF THE INVENTION

The present invention provides experimentally-generated cold-adaptedequine influenza viruses, reassortant influenza A viruses that compriseat least one genome segment of an equine influenza virus generated bycold-adaptation such that the equine influenza virus genome segmentconfers at least one identifying phenotype of a cold-adapted equineinfluenza virus on the reassortant virus, and genetically-engineeredequine influenza viruses, produced through reverse genetics, whichcomprise at least one identifying phenotype of a cold-adapted equineinfluenza virus. Identifying phenotypes include cold-adaptation,temperature sensitivity, dominant interference, and attenuation. Theinvention further provides a therapeutic composition to protect ananimal against disease caused by an influenza A virus, where thetherapeutic composition includes a cold-adapted equine influenza virus areassortant influenza A virus, or a genetically-engineered equineinfluenza virus of the present invention. Also provided is a method toprotect an animal from diseases caused by an influenza A virus whichincludes the administration of such a therapeutic composition. Alsoprovided are methods to produce a cold-adapted equine influenza virus,and methods to produce a reassortant influenza A virus which comprisesat least one genome segment of a cold-adapted equine influenza virus,where the equine influenza genome segment confers on the reassortantvirus at least one identifying phenotype of the cold-adapted equineinfluenza virus.

A cold-adapted equine influenza virus is one that replicates inembryonated chicken eggs at a temperature ranging from about 26° C. toabout 30° C. Preferably, a cold-adapted equine influenza virus,reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention is attenuated, such that itwill not cause disease in a healthy animal.

In one embodiment, a cold-adapted equine influenza virus, reassortantinfluenza A virus, or genetically-engineered equine influenza virus ofthe present invention is also temperature sensitive, such that the virusreplicates in embryonated chicken eggs at a temperature ranging fromabout 26° C. to about 30° C., forms plaques in tissue culture cells at apermissive temperature of about 34° C., but does not form plaques intissue culture cells at a non-permissive temperature of about 39° C.

In one embodiment, such a temperature sensitive virus comprises twomutations: a first mutation that inhibits plaque formation at atemperature of about 39° C., that mutation co-segregating with thegenome segment that encodes the viral nucleoprotein gene; and a secondmutation that inhibits all viral protein synthesis at a temperature ofabout 39° C.

In another embodiment, a cold-adapted, temperature sensitive equineinfluenza virus of the present invention replicates in embryonatedchicken eggs at a temperature ranging from about 26° C. to about 30° C.,forms plaques in tissue culture cells at a permissive temperature ofabout 34° C., but does not form plaques in tissue culture cells orexpress late viral proteins at a non-permissive temperature of about 37°C.

Typically, a cold-adapted equine influenza virus of the presentinvention is produced by passaging a wild-type equine influenza virusone or more times, and then selecting viruses that stably grow andreplicate at a reduced temperature. A cold-adapted equine influenzavirus produced thereby includes, in certain embodiments, a dominantinterference phenotype, that is, the virus, when co-infected with aparental equine influenza virus or heterologous wild-type influenza Avirus, will inhibit the growth of that virus.

Examples of cold-adapted equine influenza viruses of the presentinvention include EIV-P821, identified by accession No. ATCC VR ______;EIV-P824, identified by accession No. ATCC VR ______; EIV-MSV+5,identified by accession No. ATCC VR ______; and progeny of such viruses.

Therapeutic compositions of the present invention include from about 10⁵TCID₅₀ units to about 10⁸ TCID₅₀ units, and preferably about 2×10⁶TCID₅₀ units, of a cold-adapted equine influenza virus, reassortantinfluenza A virus, or genetically-engineered equine influenza virus ofthe present invention.

The present invention also includes a method to protect an animal fromdisease caused by an influenza A virus, which includes the step ofadministering to the animal a therapeutic composition including acold-adapted equine influenza virus, a reassortant influenza A virus, ora genetically-engineered equine influenza virus of the presentinvention. Preferred animals to protect include equids, with horses andponies being particularly preferred.

Yet another embodiment of the present invention is a method to generatea cold-adapted equine influenza virus. The method includes the steps ofpassaging a wild-type equine influenza virus; and selecting viruses thatgrow at a reduced temperature. In one embodiment, the method includesrepeating the passaging and selection steps one or more times, whileprogressively reducing the temperature. Passaging of equine influenzavirus preferably takes place in embryonated chicken eggs.

Another embodiment is an method to produce a reassortant influenza Avirus through genetic reassortment of the genome segments of a donorcold-adapted equine influenza virus of the present invention with thegenome segments of a recipient influenza A virus. Reassortant influenzaA viruses of the present invention are produced by a method thatincludes the steps of: (a) mixing the genome segments of a donorcold-adapted equine influenza virus with the genome segments of arecipient influenza A virus, and (b) selecting viruses which include atleast one identifying phenotype of the donor equine influenza virus.Identifying phenotypes include cold-adaptation, temperature sensitivity,dominant interference, and attenuation. Preferably, such reassortantviruses at least include the attenuation phenotype of the donor virus. Atypical reassortant virus will have the antigenicity of the recipientvirus, that is, it will retain the hemagglutinin (HA) and neuraminidase(NA) phenotypes of the recipient virus.

The present invention further provides methods to propagate cold-adaptedequine influenza viruses or reassortant influenza A viruses of thepresent invention. These methods include propagation in embryonatedchicken eggs or in tissue culture cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides experimentally-generated cold-adaptedequine influenza viruses comprising certain defined phenotypes, whichare disclosed herein. It is to be noted that the term “a” or “an”entity, refers to one or more of that entity; for example, “acold-adapted equine influenza virus” can include one or morecold-adapted equine influenza viruses. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising,” “including,” and“having” can be used interchangeably. Furthermore, an item “selectedfrom the group consisting of” refers to one or more of the items in thatgroup, including combinations thereof.

A cold-adapted equine influenza virus of the present invention is avirus that has been generated in the laboratory, and as such, is not avirus as occurs in nature. Since the present invention also includesthose viruses having the identifying phenotypes of such a cold-adaptedequine influenza virus, an equine influenza virus isolated from amixture of naturally-occurring viruses, i.e., removed from its naturalmilieu, but having the claimed phenotypes, is included in the presentinvention. A cold-adapted equine influenza virus of the presentinvention does not require any specific level of purity. For example, acold-adapted equine influenza virus grown in embryonated chicken eggsmay be in a mixture with the allantoic fluid (AF), and a cold-adaptedequine influenza virus grown in tissue culture cells may be in a mixturewith disrupted cells and tissue culture medium.

As used herein, an “equine influenza virus” is an influenza virus thatinfects and grows in equids, e.g., horses or ponies. As used herein,“growth” of a virus denotes the ability of the virus to reproduce or“replicate” itself in a permissive host cell. As such, the terms,“growth of a virus” and “replication of a virus” are usedinterchangeably herein. Growth or replication of a virus in a particularhost cell can be demonstrated and measured by standard methodswell-known to those skilled in the art of virology. For example, samplescontaining infectious virus, e.g., as contained in nasopharyngealsecretions from an infected horse, are tested for their ability to causecytopathic effect (CPE), e.g., virus plaques, in tissue culture cells.Infectious virus may also be detected by inoculation of a sample intothe allantoic cavity of embryonated chicken eggs, and then testing theAF of eggs thus inoculated for its ability to agglutinate red bloodcells, i.e., cause hemagglutination, due to the presence of theinfluenza virus hemagglutinin (HA) protein in the AF.

Naturally-occurring, i.e., wild-type, equine influenza viruses replicatewell at a temperature from about 34° C. to about 39° C. For example,wild-type equine influenza virus replicates in embryonated chicken eggsat a temperature of about 34° C., and replicates in tissue culture cellsat a temperature from about 34° C. to about 39° C. As used herein, a“cold-adapted” equine influenza virus is an equine influenza virus thathas been adapted to grow at a temperature lower than the optimal growthtemperature for equine influenza virus. One example of a cold-adaptedequine influenza virus of the present invention is a virus thatreplicates in embryonated chicken eggs at a temperature of about 30° C.A preferred cold-adapted equine influenza virus of the present inventionreplicates in embryonated chicken eggs at a temperature of about 28° C.Another preferred cold-adapted equine influenza virus of the presentinvention replicates in embryonated chicken eggs at a temperature ofabout 26° C. In general, preferred cold-adapted equine influenza virusesof the present invention replicate in embryonated chicken eggs at atemperature ranging from about 26° C. to about 30° C., i.e., at a rangeof temperatures at which a wild-type virus will grow poorly or not atall. It should be noted that the ability of such viruses to replicatewithin that temperature range does not preclude their ability to alsoreplicate at higher or lower temperatures. For example, one embodimentis a cold-adapted equine influenza virus that replicates in embryonatedchicken eggs at a temperature of about 26° C., but also replicates intissue culture cells at a temperature of about 34° C. As with wild-typeequine influenza viruses, cold-adapted equine influenza viruses of thepresent invention generally form plaques in tissue culture cells, forexample Madin Darby Canine Kidney Cells (MDCK) at a temperature of about34° C. Examples of suitable and preferred cold-adapted equine influenzaviruses of the present invention are disclosed herein.

One embodiment of the present invention is a cold-adapted equineinfluenza virus that is produced by a method which includes passaging awild-type equine influenza virus, and then selecting viruses that growat a reduced temperature. Cold-adapted equine influenza viruses of thepresent invention can be produced, for example, by sequentiallypassaging a wild-type equine influenza virus in embryonated chicken eggsat progressively lower temperatures, thereby selecting for certainmembers of the virus mixture which stably replicate at the reducedtemperature. An example of a passaging procedure is disclosed in detailin the Examples section. During the passaging procedure, one or moremutations appear in certain of the single-stranded RNA segmentscomprising the influenza virus genome, which alter the genotype, i.e.,the primary nucleotide sequence of those RNA segments. As used herein, a“mutation” is an alteration of the primary nucleotide sequence of anygiven RNA segment making up an influenza virus genome. Examples ofmutations include substitution of one or more nucleotides, deletion ofone or more nucleotides, insertion of one or more nucleotides, orinversion of a stretch of two or more nucleotides. By selecting forthose members of the virus mixture that stably replicate at a reducedtemperature, a virus with a cold-adaptation phenotype is selected. Asused herein, a “phenotype” is an observable or measurable characteristicof a biological entity such as a cell or a virus, where the observedcharacteristic is attributable to a specific genetic configuration ofthat biological entity, i.e., a certain genotype. As such, acold-adaptation phenotype is the result of one or more mutations in thevirus genome. As used herein, the terms “a mutation,” “a genome,” “agenotype,” or “a phenotype” refer to one or more, or at least onemutation, genome, genotype, or phenotype, respectively.

Additional, observable phenotypes in a cold-adapted equine influenzavirus may occur, and will generally be the result of one or moreadditional mutations in the genome of such a virus. For example, acold-adapted equine influenza virus of the present invention may, inaddition, be attenuated, exhibit dominant interference, and/or betemperature sensitive.

In one embodiment, a cold-adapted equine influenza virus of the presentinvention has a phenotype characterized by attenuation. A cold-adaptedequine influenza virus is “attenuated,” when administration of the virusto an equine influenza virus-susceptible animal results in reduced orabsent clinical signs in that animal, compared to clinical signsobserved in animals that are infected with wild-type equine influenzavirus. For example, an animal infected with wild-type equine influenzavirus will display fever, sneezing, coughing, depression, and nasaldischarges. In contrast, an animal administered an attenuated,cold-adapted equine influenza virus of the present invention willdisplay minimal or no, i.e., undetectable, clinical disease signs.

In another embodiment, a cold-adapted equine influenza virus of thepresent invention comprises a temperature sensitive phenotype. As usedherein, a temperature sensitive cold-adapted equine influenza virusreplicates at reduced temperatures, but no longer replicates or formsplaques in tissue culture cells at certain higher growth temperatures atwhich the wild-type virus will replicate and form plaques. While notbeing bound by theory, it is believed that replication of equineinfluenza viruses with a temperature sensitive phenotype is largelyrestricted to the cool passages of the upper respiratory tract, and doesnot replicate efficiently in the lower respiratory tract, where thevirus is more prone to cause disease symptoms. A temperature at which atemperature sensitive virus will grow is referred to herein as a“permissive” temperature for that temperature sensitive virus, and ahigher temperature at which the temperature sensitive virus will notgrow, but at which a corresponding wild-type virus will grow, isreferred to herein as a “non-permissive” temperature for thattemperature sensitive virus. For example, certain temperature sensitivecold-adapted equine influenza viruses of the present invention replicatein embryonated chicken eggs at a temperature at or below about 30° C.,preferably at about 28° C. or about 26° C., and will form plaques intissue culture cells at a permissive temperature of about 34° C., butwill not form plaques in tissue culture cells at a non-permissivetemperature of about 39° C. Other temperature sensitive cold-adaptedequine influenza viruses of the present invention replicate inembryonated chicken eggs at a temperature at or below about 30° C.,preferably at about 28° C. or about 26° C., and will form plaques intissue culture cells at a permissive temperature of about 34° C., butwill not form plaques in tissue culture cells at a non-permissivetemperature of about 37° C.

Certain cold-adapted equine influenza viruses of the present inventionhave a dominant interference phenotype; that is, they dominate aninfection when co-infected into cells with another influenza A virus,thereby impairing the growth of that other virus. For example, when acold-adapted equine influenza virus of the present invention, having adominant interference phenotype, is co-infected into MDCK cells with thewild-type parental equine influenza virus, A/equine/Kentucky/1/91(H3N8), growth of the parental virus is impaired. Thus, in an animalthat has recently been exposed to, or may be soon exposed to, a virulentinfluenza virus, i.e., an influenza virus that causes disease symptoms,administration of a therapeutic composition comprising a cold-adaptedequine influenza virus having a dominant interference phenotype into theupper respiratory tract of that animal will impair the growth of thevirulent virus, thereby ameliorating or reducing disease in that animal,even in the absence of an immune response to the virulent virus.

Dominant interference of a cold-adapted equine influenza virus having atemperature sensitive phenotype can be measured by standard virologicalmethods. For example, separate monolayers of MDCK cells can be infectedwith (a) a virulent wild-type influenza A virus, (b) a temperaturesensitive, cold-adapted equine influenza virus, and (c) both viruses ina co-infection, with all infections done at multiplicities of infection(MOI) of about 2 plaque forming units (pfu) per cell. After infection,the virus yields from the various infected cells are measured byduplicate plaque assays performed at the permissive temperature for thecold-adapted equine influenza virus and at the non-permissivetemperature of that virus. A cold adapted equine influenza virus havinga temperature sensitive phenotype is unable to form plaques at itsnon-permissive temperature, while the wild-type virus is able to formplaques at both the permissive and non-permissive temperatures. Thus itis possible to measure the growth of the wild-type virus in the presenceof the cold adapted virus by comparing the virus yield at thenon-permissive temperature of the cells singly infected with wild-typevirus to the yield at the non-permissive temperature of the wild-typevirus in doubly infected cells.

Cold-adapted equine influenza viruses of the present invention arecharacterized primarily by one or more of the following identifyingphenotypes: cold-adaptation, temperature sensitivity, dominantinterference, and/or attenuation. As used herein, the phrase “an equineinfluenza virus comprises the identifying phenotype(s) ofcold-adaptation, temperature sensitivity, dominant interference, and/orattenuation” refers to a virus having such a phenotype(s). Examples ofsuch viruses include, but are not limited to, EIV-P821, identified byaccession No. ATCC VR ______, EIV-P824, identified by accession No. ATCCVR ______, and EIV-MSV+5, identified by accession No. ATCC VR ______, aswell as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4.Production of such viruses is described in the examples. For example,cold-adapted equine influenza virus EIV-P821 is characterized by, i.e.,has the identifying phenotypes of, (a) cold-adaptation, e.g., itsability to replicate in embryonated chicken eggs at a temperature ofabout 26° C.; (b) temperature sensitivity, e.g., its inability to formplaques in tissue culture cells and to express late gene products at anon-permissive temperature of about 37° C., and its inability to formplaques in tissue culture cells and to synthesize any viral proteins ata non-permissive temperature of about 39° C.; (c) its attenuation uponadministration to an equine influenza virus-susceptible animal; and (d)dominant interference, e.g., its ability, when co-infected into a cellwith a wild-type influenza A virus, to interfere with the growth of thatwild-type virus. Similarly, cold-adapted equine influenza virus EIV-P824is characterized by (a) cold adaptation, e.g., its ability to replicatein embryonated chicken eggs at a temperature of about 28° C.; (b)temperature sensitivity, e.g., its inability to form plaques in tissueculture cells at a non-permissive temperature of about 39° C.; and (c)dominant interference, e.g., its ability, when co-infected into a cellwith a wild-type influenza A virus, to interfere with the growth of thatwild-type virus. In another example, cold-adapted equine influenza virusEIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its ability toreplicate in embryonated chicken eggs at a temperature of about 26° C.;(b) temperature sensitivity, e.g., its inability to form plaques intissue culture cells at a non-permissive temperature of about 39° C.;and (c) its attenuation upon administration to an equine influenzavirus-susceptible animal.

In certain cases, the RNA segment upon which one or more mutationsassociated with a certain phenotype occur may be determined throughreassortment analysis by standard methods, as disclosed herein. In oneembodiment, a cold-adapted equine influenza virus of the presentinvention comprises a temperature sensitive phenotype that correlateswith at least two mutations in the genome of that virus. In thisembodiment, one of the two mutations, localized by reassortment analysisas disclosed herein, inhibits, i.e., blocks or prevents, the ability ofthe virus to form plaques in tissue culture cells at a non-permissivetemperature of about 39° C. This mutation co-segregates with the segmentof the equine influenza virus genome that encodes the nucleoprotein (NP)gene of the virus, i.e., the mutation is located on the same RNA segmentas the NP gene. In this embodiment, the second mutation inhibits allprotein synthesis at a non-permissive temperature of about 39° C. Assuch, at the non-permissive temperature, the virus genome is incapableof expressing any viral proteins. Examples of cold-adapted equineinfluenza viruses possessing these characteristics are EIV-P821 and EIVMSV+5. EIV-P821 was generated by serial passaging of a wild-type equineinfluenza virus in embryonated chicken eggs by methods described inExample 1A. EIV-MSV+5 was derived by further serial passaging ofEIV-P821, as described in Example 1E.

Furthermore, a cold-adapted, temperature sensitive equine influenzavirus comprising the two mutations which inhibit plaque formation andviral protein synthesis at a non-permissive temperature of about 39° C.can comprise one or more additional mutations, which inhibit the virus'ability to synthesize late gene products and to form plaques in tissueculture cells at a non-permissive temperature of about 37° C. An exampleof a cold-adapted equine influenza virus possessing thesecharacteristics is EIV-P821. This virus isolate replicates inembryonated chicken eggs at a temperature of about 26° C., and does notform plaques or express any viral proteins at a temperature of about 39°C. Furthermore, EIV-P821 does not form plaques on MDCK cells at anon-permissive temperature of about 37° C., and at this temperature,late gene expression is inhibited in such a way that late proteins arenot produced, i.e., normal levels of NP protein are synthesized, reducedor undetectable levels of M1 or HA proteins are synthesized, andenhanced levels of the polymerase proteins are synthesized. Since thisphenotype is typified by differential viral protein synthesis, it isdistinct from the protein synthesis phenotype seen at a non-permissivetemperature of about 39° C., which is typified by the inhibition ofsynthesis of all viral proteins.

Pursuant to 37 CFR § 1.802 (a-c), cold-adapted equine influenza viruses,designated herein as FIV-P821, an EIV-P824 were deposited with theAmerican Type Culture Collection (ATCC, 10801 University Boulevard,Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC AccessionNos. ATCC VR-______, and ATCC VR-______, respectively, on Jul. 11, 1998.Cold-adapted equine influenza virus EIV-MSV+5 was deposited with theATCC as ATCC Accession No. ATCC VR-______ on Aug. 3, 1998. Pursuant to37 CFR§ 1.806, the deposits are made for a term of at least thirty (30)years and at least five (5) years after the most recent request for thefurnishing of a sample of the deposit was received by the depository.Pursuant to 37 CFR § 1.808 (a)(2), all restrictions imposed by thedepositor on the availability to the public will be irrevocably removedupon the granting of the patent.

Preferred cold-adapted equine influenza viruses of the present inventionhave the identifying phenotypes of EIV-P821, EIV-P824, and EIV-MSV+5.Particularly preferred cold-adapted equine influenza viruses includeEIV-P821, EIV-P824, EIV-MSV+5, and progeny of these viruses. As usedherein, “progeny” are “offspring,” and as such can slightly alteredphenotypes compared to the parent virus, but retain identifyingphenotypes of the parent virus, for example, cold-adaptation,temperature sensitivity, dominant interference, or attenuation. Forexample, cold-adapted equine influenza virus EIV-MSV+5 is a “progeny” ofcold-adapted equine influenza virus EIV-P821. “Progeny” also includereassortant influenza A viruses that comprise one or more identifyingphenotypes of the donor parent virus.

Reassortant influenza A viruses of the present invention are produced bygenetic reassortment of the genome segments of a donor cold-adaptedequine influenza virus of the present invention with the genome segmentsof a recipient influenza A virus, and then selecting a reassortant virusthat derives at least one of its eight RNA genome segments from thedonor virus, such that the reassortant virus acquires at least oneidentifying phenotype of the donor cold-adapted equine influenza virus.Identifying phenotypes include cold-adaptation, temperature sensitivity,attenuation, and dominant interference. Preferably, reassortantinfluenza A viruses of the present invention derive at least theattenuation phenotype of the donor virus. Methods to isolate reassortantinfluenza viruses are well known to those skilled in the art of virologyand are disclosed, for example, in Fields, et al., 1996, FieldsVirology, 3d ed., Lippincott-Raven; and Palese, et al., 1976, J. Virol.,17, 876-884. Fields, et al., ibid. and Palese, et al., ibid.

A suitable donor equine influenza virus is a cold-adapted equineinfluenza virus of the present invention, for example, EIV-P821,identified by accession No. ATCC VR ______, EIV-P824, identified byaccession No. ATCC VR ______, or EIV-MSV+5, identified by accession No.ATCC VR ______. A suitable recipient influenza A virus can be anotherequine influenza virus, for example a Eurasian subtype 2 equineinfluenza virus such as A/equine/Suffolk/89 (H3N8) or a subtype 1 equineinfluenza virus such as A/Prague/1/56 (H7N7). A recipient influenza Avirus can also be any influenza A virus capable of forming a reassortantvirus with a donor cold-adapted equine influenza virus. Examples of suchinfluenza A viruses include, but are not limited to, human influenzaviruses such as A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1),A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses suchas A/Swine/Iowa/15/30 (H1N1); and avian viruses such as A/mallard/NewYork/6750/78 (H2N2) and A/chicken/Hong Kong/258/97 (H5N1). A reassortantvirus of the present invention can include any combination of donor andrecipient gene segments, as long as the resulting reassortant viruspossesses at least one identifying phenotype of the donor virus.

One example of a reassortant virus of the present invention is a “6+2”reassortant virus, in which the six “internal gene segments,” i.e.,those comprising the NP, PB2, PB1, PA, M, and NS genes, are derived fromthe donor cold-adapted equine influenza virus genome, and the two“external gene segments,” i.e., those comprising the HA and NA genes,are derived from the recipient influenza A virus. A resultant virus thusproduced has the attenuated, cold-adapted, temperature sensitive, and/ordominant interference phenotypes of the donor cold-adapted equineinfluenza virus, but the antigenicity of the recipient strain.

In yet another embodiment, a cold-adapted equine influenza virus of thepresent invention can be produced through recombinant means. In thisapproach, one or more specific mutations, associated with identifiedcold-adaptation, attenuation, temperature sensitivity, or dominantinterference phenotypes, are identified and are introduced back into awild-type equine influenza virus strain using a reverse geneticsapproach. Reverse genetics entails using RNA polymerase complexesisolated from influenza virus-infected cells to transcribe artificialinfluenza virus genome segments containing the mutation(s),incorporating the synthesized RNA segment(s) into virus particles usinga helper virus, and then selecting for viruses containing the desiredchanges. Reverse genetics methods for influenza viruses are described,for example, in Enami, et al., 1990, Proc. Natl. Acad. Sci. 87,3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et al., issuedNov. 26, 1996. This approach allows one skilled in the art to produceadditional cold-adapted equine influenza viruses of the presentinvention without the need to go through the lengthy cold-adaptationprocess, and the process of selecting mutants both in vitro and in vivowith the desired virus phenotype.

A cold-adapted equine influenza virus of the present invention may bepropagated by standard virological methods well-known to those skilledin the art, examples of which are disclosed herein. For example, acold-adapted equine influenza virus can be grown in embryonated chickeneggs or in eukaryotic tissue culture cells. Suitable continuouseukaryotic cell lines upon which to grow a cold-adapted equine influenzavirus of the present invention include those that support growth ofinfluenza viruses, for example, MDCK cells. Other suitable cells uponwhich to grow a cold-adapted equine influenza virus of the presentinvention include, but are not limited to, primary kidney cell culturesof monkey, calf, hamster or chicken.

In one embodiment, the present invention provides a therapeuticcomposition to protect an animal against disease caused by an influenzaA virus, where the therapeutic composition includes either acold-adapted equine influenza virus or a reassortant influenza A viruscomprising at least one genome segment of an equine influenza virusgenerated by cold-adaptation, wherein the equine influenza virus genomesegment confers at least one identifying phenotype of the cold-adaptedequine influenza virus. In addition, a therapeutic composition of thepresent invention can include an equine influenza virus that has beengenetically engineered to comprise one or more mutations, where thosemutations have been identified to confer a certain identifying phenotypeon a cold-adapted equine influenza virus of the present invention. Asused herein, the phrase “disease caused by an influenza A virus” refersto the clinical manifestations observed in an animal which has beeninfected with a virulent influenza A virus. Examples of such clinicalmanifestations include, but are not limited to, fever, sneezing,coughing, nasal discharge, rales, anorexia and depression. In addition,the phrase “disease caused by an influenza A virus” is defined herein toinclude shedding of virulent virus by the infected animal. Verificationthat clinical manifestations observed in an animal correlate withinfection by virulent equine influenza virus may be made by severalmethods, including the detection of a specific antibody and/or T-cellresponses to equine influenza virus in the animal. Preferably,verification that clinical manifestations observed in an animalcorrelate with infection by a virulent influenza A virus is made by theisolation of the virus from the afflicted animal, for example, byswabbing the nasopharyngeal cavity of that animal for virus-containingsecretions. Verification of virus isolation may be made by the detectionof CPE in tissue culture cells inoculated with the isolated secretions,by inoculation of the isolated secretions into embryonated chicken eggs,where virus replication is detected by the ability of AF from theinoculated eggs to agglutinate erythrocytes, suggesting the presence ofthe influenza virus hemagglutinin protein, or by use of a commerciallyavailable diagnostic test, for example, the Directigen® FLU A test.

As used herein, the term “to protect” includes, for example, to preventor to treat influenza A virus infection in the subject animal. As such,a therapeutic composition of the present invention can be used, forexample, as a prophylactic vaccine to protect a subject animal frominfluenza disease by administering the therapeutic composition to thatanimal at some time prior to that animal's exposure to the virulentvirus.

A therapeutic composition of the present invention, comprising acold-adapted equine influenza virus having a dominant interferencephenotype, can also be used to treat an animal that has been recentlyinfected with virulent influenza A virus or is likely to be subsequentlyexposed in a few days, such that the therapeutic composition immediatelyinterferes with the growth of the virulent virus, prior to the animal'sproduction of antibodies to the virulent virus. A therapeuticcomposition comprising a cold-adapted equine influenza virus having adominant interference phenotype may be effectively administered prior tosubsequent exposure for a length of time corresponding to theapproximate length of time that a cold-adapted equine influenza virus ofthe present invention will replicate in the upper respiratory tract of atreated animal, for example, up to about seven days. A therapeuticcomposition comprising a cold-adapted equine influenza virus having adominant interference phenotype may be effectively administeredfollowing exposure to virulent equine influenza virus for a length oftime corresponding to the time required for an infected animal to showdisease symptoms, for example, up to about two days.

Therapeutic compositions of the present invention can be administered toany animal susceptible to influenza virus disease, for example, humans,swine, horses and other equids, aquatic birds, domestic and game fowl,seals, mink, and whales. Preferably, a therapeutic composition of thepresent invention is administered equids. Even more preferably, atherapeutic composition of the present invention is administered to ahorse, to protect against equine influenza disease.

Current vaccines available to protect horses against equine influenzavirus disease are not effective in protecting young foals, most likelybecause they cannot overcome the maternal antibody present in theseyoung animals, and often, vaccination at an early age, for example 3months of age, can lead to tolerance rather than immunity. In oneembodiment, and in contrast to existing equine influenza virus vaccines,a therapeutic composition comprising a cold-adapted equine influenzavirus of the present invention apparently can produce immunity in younganimals. As such, a therapeutic composition of the present invention canbe safely and effectively administered to young foals, as young as about3 months of age, to protect against equine influenza disease without theinduction of tolerance.

In one embodiment, a therapeutic composition of the present inventioncan be multivalent. For example, it can protect an animal from more thanone strain of influenza A virus by providing a combination of one ormore cold-adapted equine influenza viruses of the present invention, oneor more reassortant influenza A viruses, and/or one or moregenetically-engineered equine influenza viruses of the presentinvention. Multivalent therapeutic compositions can include at least twocold-adapted equine influenza viruses, e.g., against North Americansubtype-2 virus isolates such as A/equine/Kentucky/1/91 (H1N8), andEurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); orone or more subtype-2 virus isolates and a subtype-1 virus isolate suchas A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeuticcomposition of the present invention can include a cold-adapted equineinfluenza virus and a reassortant influenza A virus of the presentinvention, or two reassortant influenza A viruses of the presentinvention. A multivalent therapeutic composition of the presentinvention can also contain one or more formulations to protect againstone or more other infectious agents in addition to influenza A virus.Such other infectious agents include, but not limited to: viruses;bacteria; fungi and fungal-related microorganisms; and parasites.Preferable multivalent therapeutic compositions include, but are notlimited to, a cold-adapted equine influenza virus, reassortant influenzaA virus, or genetically-engineered equine influenza virus of the presentinvention plus one or more compositions protective against one or moreother infectious agents that afflict horses. Suitable infectious agentsto protect against include, but are not limited to, equine infectiousanemia virus, equine herpes virus, eastern, western, or Venezuelanequine encephalitis virus, tetanus, Streptococcus equi, and Ehrlichiaresticii.

A therapeutic composition of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Excipients can also contain minor amounts of additives,such as substances that enhance isotonicity and chemical or biologicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer, and Tris buffer, while examples of stabilizers include A1/A2stabilizer, available from Diamond Animal Health, Des Moines, Iowa.Standard formulations can either be liquids or solids which can be takenup in a suitable liquid as a suspension or solution for administrationto an animal. In one embodiment, a non-liquid formulation may comprisethe excipient salts, buffers, stabilizers, etc., to which sterile wateror saline can be added prior to administration.

A therapeutic composition of the present invention may also include oneor more adjuvants or carriers. Adjuvants are typically substances thatenhance the immune response of an animal to a specific antigen, andcarriers include those compounds that increase the half-life of atherapeutic composition in the treated animal. One advantage of atherapeutic composition comprising a cold-adapted equine influenza virusor a reassortant influenza A virus of the present invention is thatadjuvants and carriers are not required to produce an efficaciousvaccine. Furthermore, in many cases known to those skilled in the art,the advantages of a therapeutic composition of the present inventionwould be hindered by the use of some adjuvants or carriers. However, itshould be noted that use of adjuvants or carriers is not precluded bythe present invention.

Therapeutic compositions of the present invention include an amount of acold-adapted equine influenza virus that is sufficient to protect ananimal from challenge with virulent equine influenza virus. In oneembodiment, a therapeutic composition of the present invention caninclude an amount of a cold-adapted equine influenza virus ranging fromabout 10⁵ tissue culture infectious dose-50 (TCID₅₀) units of virus toabout 10⁸ TCTD₅₀ units of virus. As used herein, a “TCID₅₀ unit” isamount of a virus which results in cytopathic effect in 50% of thosecell cultures infected. Methods to measure and calculate TCID₅₀ areknown to those skilled in the art and are available, for example, inReed and Muench, 1938, Am. J. of Hyg. 27, 493-497. A preferredtherapeutic composition of the present invention comprises from about10⁶ TCID₅₀ units to about 10⁷ TCID₅₀ units of a cold-adapted equineinfluenza virus or reassortant influenza A virus of the presentinvention. Even more preferred is a therapeutic composition comprisingabout 2×10⁶ TCID₅₀ units of a cold-adapted equine influenza virus orreassortant influenza A virus of the present invention.

The present invention also includes methods to protect an animal againstdisease caused by an influenza A virus comprising administering to theanimal a therapeutic composition of the present invention. Preferred arethose methods which protect an equid against disease caused by equineinfluenza virus, where those methods comprise administering to the equida cold-adapted equine influenza virus. Acceptable protocols toadminister therapeutic compositions in an effective manner includeindividual dose size, number of doses, frequency of dose administration,and mode of administration. Determination of such protocols can beaccomplished by those skilled in the art, and examples are disclosedherein.

A preferable method to protect an animal against disease caused by aninfluenza A virus includes administering to that animal a single dose ofa therapeutic composition comprising a cold-adapted equine influenzavirus, a reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention. A suitable single dose is adose that is capable of protecting an animal from disease whenadministered one or more times over a suitable time period. The methodof the present invention may also include administering subsequent, orbooster doses of a therapeutic composition. Booster administrations canbe given from about 2 weeks to several years after the originaladministration. Booster administrations preferably are administered whenthe immune response of the animal becomes insufficient to protect theanimal from disease. Examples of suitable and preferred dosage schedulesare disclosed in the Examples section.

A therapeutic composition of the present invention can be administeredto an animal by a variety of means, such that the virus will enter andreplicate in the mucosal cells in the upper respiratory tract of thetreated animal. Such means include, but are not limited to, intranasaladministration, oral administration, and intraocular administration.Since influenza viruses naturally infect the mucosa of the upperrespiratory tract, a preferred method to administer a therapeuticcomposition of the present invention is by intranasal administration.Such administration may be accomplished by use of a syringe fitted withcannula, or by use of a nebulizer fitted over the nose and mouth of theanimal to be vaccinated.

The efficacy of a therapeutic composition of the present invention toprotect an animal against disease caused by influenza A virus can betested in a variety of ways including, but not limited to, detection ofantibodies by, for example, hemagglutination inhibition (HAI) tests,detection of cellular immunity within the treated animal, or challengeof the treated animal with virulent equine influenza virus to determinewhether the treated animal is resistant to the development of disease.In addition, efficacy of a therapeutic composition of the presentinvention comprising a cold-adapted equine influenza virus having adominant interference phenotype to ameliorate or reduce disease symptomsin an animal previously inoculated or susceptible to inoculation with avirulent, wild-type equine influenza virus can be tested by screeningfor the reduction or absence of disease symptoms in the treated animal.

The present invention also includes methods to produce a therapeuticcomposition of the present invention. Suitable and preferred methods formaking a therapeutic composition of the present invention are disclosedherein. Pertinent steps involved in producing one type of therapeuticcomposition of the present invention, i.e., a cold-adapted equineinfluenza virus, include (a) passaging a wild-type equine influenzavirus in vitro, for example, in embryonated chicken eggs; (b) selectingviruses that grow at a reduced temperature; (c) repeating the passagingand selection steps one or more times, at progressively lowertemperatures, until virus populations are selected which stably grow atthe desired lower temperature; and (d) mixing the resulting viruspreparation with suitable excipients.

The pertinent steps involved in producing another type of therapeuticcomposition of the present invention, i.e., a reassortant influenza Avirus having at least one genome segment of an equine influenza virusgenerated by adaptation, includes the steps of (a) mixing the genomesegments of a donor cold-adapted equine influenza virus, whichpreferably also has the phenotypes of attenuation, temperaturesensitivity, or dominant interference, with the genome segments of arecipient influenza A virus, and (b) selecting reassortant viruses thathave at least one identifying phenotype of the donor equine influenzavirus. Identifying phenotypes to select for include attenuation,cold-adaptation, temperature sensitivity, and dominant interference.Methods to screen for these phenotypes are well known to those skilledin the art, and are disclosed herein. It is preferable to screen forviruses that at least have the phenotype of attenuation.

Using this method to generate a reassortant influenza A virus having atleast one genome segment of a equine influenza virus generated bycold-adaptation, one type of reassortant virus to select for is a “6+2”reassortant, where the six “internal gene segments,” i.e., those codingfor the NP, PB2, PB1, PA, M, and NS genes, are derived from the donorcold-adapted equine influenza virus genome, and the two “external genesegments,” i.e., those coding for the HA and NA genes, are derived fromthe recipient influenza A virus. A resultant virus thus produced canhave the cold-adapted, attenuated, temperature sensitive, and/orinterference phenotypes of the donor cold-adapted equine influenzavirus, but the antigenicity of the recipient strain.

The present invention includes nucleic acid molecules isolated fromequine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8),and cold-adapted equine influenza virus EIV-P821.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified.

The present invention includes nucleic acid molecules encoding wild-typeand cold-adapted equine influenza virus proteins. Nucleic acid moleculesof the present invention can be prepared by methods known to one skilledin the art. Proteins of the present invention can be prepared by methodsknown to one skilled in the art, i.e., recombinant DNA technology.Preferred nucleic acid molecules have coding strands comprising nucleicacid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:25, and/or a complement thereof.Complements are defined as two single strands of nucleic acid in whichthe nucleotide sequence is such that they will hybridize as a result ofbase pairing throughout their full length. Given a nucleotide sequence,one of ordinary skill in the art can deduce the complement.

Preferred nucleic acid molecules encoding equine influenza M proteinsare nei_(wt)M₁₀₂₃, nei_(wt1)M₁₀₂₃, nei_(wt2)M₁₀₂₃, nei_(wt)M₇₅₆,nei_(wt1)M₇₅₆, nei_(wt2)M₇₅₆, nei_(ca1)M₁₀₂₃, nei_(ca2)M₁₀₂₃,nei_(ca1)M₇₅₆ and/or ne_(ca2)M₇₅₆, the coding strands of which arerepresented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ IDNO:6.

Preferred nucleic acid molecules encoding equine influenza HA proteinsare nei_(wt)HA₁₇₆₂, nei_(wt)HA₁₆₉₅, nei_(ca1)HA₁₇₆₂, nei_(ca2)HA₁₇₆₂,nei_(ca1)HA₁₆₉₅, and/or nei_(ca2)HA₁₆₉₅, the coding strands of which arerepresented by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ IDNO:12.

Preferred nucleic acid molecules encoding equine influenza PB2-Nproteins are nei_(wt)PB2-N₁₂₄₁, nei_(wt)PB2-N₁₂₁₄, nei_(ca1)PB2-N₁₂₄₁nei_(ca2)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₁₄ nei_(ca2), and/or PB2-N₁₂₁₄, thecoding strands of which are represented by SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:16, and/or SEQ ID NO:18.

Preferred nucleic acid molecules encoding equine influenza PB2-Cproteins are nei_(wt1)PB2-C₁₂₃₃, nei_(wt2)PB2-C₁₂₃₂, nei_(wt)PB2-C₁₁₉₄,nei_(ca1)PB2-C₁₂₃₂, nei_(ca2)PB2-C₁₂₃₁, and/or nei_(ca1)PB2-C₁₁₉₄, thecoding strands of which are represented by SEQ ID NO:19, SEQ ID NO:22,SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25.

The present invention includes proteins comprising SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ IDNO:20 and/or SEQ ID NO:24 as well as nucleic acid molecules encodingsuch proteins.

Preferred equine influenza M proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)M₁₀₂₃,nei_(wt1)M₁₀₂₃, nei_(wt2)M₁₀₂₃, nei_(wt)M₇₅₆, nei_(wt1)M₇₅₆,nei_(wt2)M₇₅₆, nei_(ca1)M₁₀₂₃, nei_(ca2)M₁₀₂₃, nei_(ca1)M₇₅₆, and/ornei_(ca2)M₇₅₆. Preferred equine influenza M proteins are Pei_(wt)M₂₅₂,Pei_(ca1)M₂₅₂, and/or Pei_(ca2)M₂₅₂. In one embodiment, a preferredequine influenza M protein of the present invention is encoded by SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6, and, as such, has anamino acid sequence that includes SEQ ID NO:2 and/or SEQ ID NO:5.

Preferred equine influenza HA proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)HA₁₇₆₂nei_(wt)HA₁₆₉₅, nei_(ca1)HA₁₇₆₂, nei_(ca2)HA₁₇₆₂, nei_(ca1)HA₁₆₉₅,and/or nei_(ca2)HA₁₆₉₅. Preferred equine influenza HA proteins are PPei_(wt)HA₅₆₅, Pei_(ca1)HA₅₆₅, and/or Pei_(ca2)HA₅₆₅. In one embodiment,a preferred equine influenza HA protein of the present invention isencoded by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12,and, as such, has an amino acid sequence that includes SEQ ID NO: 8and/or SEQ ID NO:11.

Preferred equine influenza PB2-N proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt)PB2-N₁₂₄₁, nei_(wt)PB2-N₁₂₁₄, nei_(ca1)PB2-N₁₂₄₁,nei_(ca2)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₁₄ nei_(ca2), and/or PB2-N₁₂₁₄.Preferred equine influenza PB2-N proteins are P_(wt)PB2-N₄₀₄,P_(ca1)PB2-N₄₀₄, and/or P_(ca2)PB2-N₄₀₄. In one embodiment, a preferredequine influenza PB2-N protein of the present invention is encoded bySEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18, and, assuch, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQID NO:17.

Preferred equine influenza PB2-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB2-C₁₂₃₃, nei_(wt2)PB2-C₁₂₃₂, nei_(wt)PB2-C₁₁₉₄,nei_(ca1)PB2-C₁₂₃₂, nei_(ca2)PB2-C₁₂₃₁, and/or nei_(ca1)PB2-C₁₁₉₄.Preferred equine influenza PB2-N proteins are P_(wt)PB2-C₃₉₈,P_(ca1)PB2-C₃₉₈, and/or P_(ca2)PB2-C₃₉₈. In one embodiment, a preferredequine influenza PB2-C protein of the present invention is encoded bySEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ IDNO:25, and, as such, has an amino acid sequence that includes SEQ IDNO:20 and/or SEQ ID NO:24.

Nucleic acid sequence SEQ ID NO:1 represents the consensus sequencededuced from the coding strand of PCR amplified nucleic acid moleculesdenoted herein as nei_(wt1)M₁₀₂₃ and nei_(wt2)M₁₀₂₃, the production ofwhich is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:4represents the deduced sequence of the coding strand of PCR amplifiednucleic acid molecules denoted herein as nei_(ca1)M₁₀₂₃ andnei_(ca2)M₁₀₂₃, the production of which is disclosed in the Examples.Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of thecoding strand of a PCR amplified nucleic acid molecule denoted herein asnei_(wt)HA₁₇₆₂, the production of which is disclosed in the Examples.Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence ofthe coding strand of PCR amplified nucleic acid molecules denoted hereinas nei_(ca1)HA₁₇₆₂ and nei_(ca2)HA₁₇₆₂, the production of which isdisclosed in the Examples. Nucleic acid sequence SEQ ID NO:13 representsthe deduced sequence of the coding strand of a PCR amplified nucleicacid molecule denoted herein as nei_(wt)PB2-N₁₂₄₁, the production ofwhich is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:16represents the deduced sequence of the coding strand of PCR amplifiednucleic acid molecules denoted herein as nei_(ca1)PB2-N₁₂₄₁ andnei_(ca2)PB2-N₁₂₄₁, the production of which is disclosed in theExamples. Nucleic acid sequence SEQ ID NO:19 represents the deducedsequence of the coding strand of a PCR amplified nucleic acid moleculedenoted herein as nei_(wt1)PB2-C₁₂₃₃, the production of which isdisclosed in the examples.

Nucleic acid sequence SEQ ID NO:22 represents the deduced sequence ofthe coding strand of a PCR amplified nucleic acid molecule denotedherein as nei_(wt2)PB2-C₁₂₃₂, the production of which is disclosed inthe examples. Nucleic acid sequence SEQ ID NO:23 represents the deducedsequence of the coding strand of a PCR amplified nucleic acid moleculedenoted herein as nei_(ca1)PB2-C₁₂₃₂, the production of which isdisclosed in the examples. Additional nucleic acid molecules, nucleicacid sequences, proteins and amino acid sequences are described in theExamples.

The present invention includes nucleic acid molecule comprising acold-adapted equine influenza virus encoding an M protein having anamino acid sequence comprising SEQ ID NO:5. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding an HA protein having anamino acid sequence comprising SEQ ID NO:11. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2-N protein having anamino acid sequence comprising SEQ ID NO:17. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2-C protein having anamino acid sequence comprising SEQ ID NO:24.

It should be noted that since nucleic acid sequencing technology is notentirely error-free, the nucleic acid sequences and amino acid sequencespresented herein represent, respectively, apparent nucleic acidsequences of nucleic acid molecules of the present invention andapparent amino acid sequences of M, HA, and PB2-N, and PB2-C proteins ofthe present invention.

Another embodiment of the present invention is an antibody thatselectively binds to an wild-type virus M, HA, PB2-N, PB2-C, PB2,protein of the present invention. Another embodiment of the presentinvention is an antibody that selectively binds to a cold-adapted virusM, HA, PB2-N, PB2-C, PB2, protein of the present invention. Preferredantibodies selectively bind to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20 and/or SEQ IDNO:24.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLE 1

This example discloses the production and phenotypic characterization ofseveral cold-adapted equine influenza viruses of the present invention.

A. Parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8)(obtained from Tom Chambers, the University of Kentucky, Lexington, Ky.)was subjected to cold-adaptation in a foreign host species, i.e.,embryonated chicken eggs, in the following manner. Embryonated, 10 or11-day old chicken eggs, available, for example, from Truslow Farms,Chestertown, Md. or from HyVac, Adel, Iowa, were inoculated with theparental equine influenza virus by injecting about 0.1 milliliter (ml)undiluted AF containing approximately 10⁶ plaque forming units (pfu) ofvirus into the allantoic cavity through a small hole punched in theshell of the egg. The holes in the eggs were sealed with nail polish andthe eggs were incubated in a humidified incubator set at the appropriatetemperature for three days. Following incubation, the eggs were candledand any non-viable eggs were discarded. AF was harvested from viableembryos by aseptically removing a portion of the egg shell, pullingaside the chorioallantoic membrane (CAM) with sterile forceps andremoving the AF with a sterile pipette. The harvested AF was frozenbetween passages. The AF was then used, either undiluted or diluted1:1000 in phosphate-buffered saline (PBS) as noted in Table 1, toinoculate a new set of eggs for a second passage, and so on. A total of69 passages were completed. Earlier passages were done at either about34° C. (passages 1-2) or about 30° C. and on subsequent passages, theincubation temperature was shifted down either to about 28° C., or toabout 26° C. In order to increase the possibility of the selection ofthe desired phenotype of a stable, attenuated virus, the initial serialpassage was expanded to included five different limbs of the serialpassage tree, A through E, as shown in Table 1. TABLE 1 Passage historyof the limbs A through E. Passage # Temperature Limb A Limb B Limb CLimb D Limb E 34° C. 1-2 1-2  1-2  1-2 1-2 30° C. 3-8 3-29 3-29  3-29 3-29 28° C. 30-33* 30-68* 30-33 30-69 26° C.  9-65 34-69* 34-65*= the infectious allantoic fluid was diluted 1:1000 in these passages

B. Virus isolates carried through the cold-adaptation proceduredescribed in section A were tested for temperature sensitivity, i.e., aphenotype in which the cold-adapted virus grows at the lower, orpermissive temperature (e.g., about 34° C.), but no longer forms plaquesat a higher, or non-permissive temperature (e.g., about 37° C. or about39° C.), as follows. At each cold-adaptation passage, the AF was titeredby plaque assay at about 34° C. Periodically, individual plaques fromthe assay were clonally isolated by excision of the plaque area andplacement of the excised agar plug in a 96-well tray containing amonolayer of MDCK cells. The 96-well trays were incubated overnight andthe yield assayed for temperature sensitivity by CPE assay in duplicate96-well trays incubated at about 34° C. and at about 39° C. The percentof the clones that scored as temperature sensitive mutants by thisassay, i.e., the number of viral plaques that grew at 34° C. but did notgrow at 39° C., divided by the total number of plaques, was calculated,and is shown in Table 2. Temperature sensitive isolates were thenevaluated for protein synthesis at the non-permissive temperature byvisualization of radiolabeled virus-synthesized proteins by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). TABLE 2 Percent ofisolated Clones that were temperature sensitive. Percent TemperatureSensitive Passage# Limb A Limb B Limb C Limb D Limb E p36  56%  66%  0% 66% 54% p46  80% 60% 75% p47 80% p48 100%  p49 100% 100% 50% p50 90%p51 100% p52 57% p62 100% 100% p65 100%  p66 100% 88%

From the clonal isolates tested for temperature sensitivity, two wereselected for further study. Clone EIV-P821 was selected from the 49thpassage of limb B and clone EIV-P824 was selected from the 48th passageof limb C as defined in Table 1. Both of these virus isolates weretemperature sensitive, with plaque formation of both isolates inhibitedat a temperature of about 39° C. At this temperature, protein synthesiswas completely inhibited by EIV-P821, but EIV-P824 exhibited normallevels of protein synthesis. In addition, plaque formation by EIV-P821was inhibited at a temperature of about 37° C., and at this temperature,late gene expression was inhibited, i.e., normal levels of NP proteinwere synthesized, reduced or no M1 or HA proteins were synthesized, andenhanced levels of the polymerase proteins were synthesized. Thephenotype observed at 37° C., being typified by differential viralprotein synthesis, was distinct from the protein synthesis phenotypeseen at about 39° C., which was typified by the inhibition of synthesisof all viral proteins. Virus EIV-P821 has been deposited with theAmerican Type Culture Collection (ATCC) under Accession No. ATCCVR-______, and virus EIV-P824 has been deposited with the ATCC underAccession No. ATCC VR-______.

C. Further characterization of the mutations in isolate EIV-P821 werecarried out by reassortment analysis, as follows. Reassortment analysisin influenza viruses allows one skilled in the art, under certaincircumstances, to correlate phenotypes of a given virus with putativemutations occurring on certain of the eight RNA segments that comprisean influenza A virus genome. This technique is described, for example,in Palese, et al., ibid. A mixed infection of EIV-P821 and an avianinfluenza virus, A/mallard/New York/6750/78 was performed as follows.MDCK cells were co-infected with EIV-P821 at a multiplicity of infection(MOT) of 2 pfu/cell and A/mallard/New York/6750/78 at an MOI of either2, 5, or 10 pfu/cell. The infected cells were incubated at a temperatureof about 34° C. The yields of the various co-infections were titered andindividual plaques were isolated at about 34° C., and the resultantclonal isolates were characterized as to whether they were able to growat about 39° C. and about 37° C., and express their genes, i.e.,synthesize viral proteins, at about 39° C., about 37° C., and about 34°C. Protein synthesis was evaluated by SDS-PAGE analysis of radiolabeledinfected-cell lysates. The HA, NP and NS-1 proteins of the two parentviruses, each of which is encoded by a separate genome segment, weredistinguishable by SDS-PAGE analysis, since these particular viralproteins, as derived from either the equine or the avian influenzavirus, migrate at different apparent molecular weights. In this way itwas possible, at least for the HA, NP, and NS-1 genes, to evaluatewhether certain phenotypes of the parent virus, e.g., the temperaturesensitive and the protein synthesis phenotypes, co-segregate with thegenome segments carrying these genes. The results of the reassortmentanalyses investigating co-segregation of a) the mutation inhibitingplaque formation, i.e., the induction of CPE, at a non-permissivetemperature of about 39° C. or b) the mutation inhibiting proteinsynthesis at a non-permissive temperature of about 39° C. with each ofthe EIV-P821 HA, NP and NS-1 proteins are shown in Tables 3 and 4,respectively. TABLE 3 Reassortment analysis of the EIV-P821 39° C.plaque formation phenotype with avian influenza virus, A/mallard/NewYork/6750/78 Gene Virus ts+¹ ts−² HA avian 26 13 equine 11 44 NP avian37 8 equine 0 49 NS-1 avian 9 8 equine 12 20¹number of clonal isolates able to induce CPE in tissue culture cells ata temperature of about 39° C.²number of clonal isolates inhibited in the ability to induce CPE intissue culture cells at a temperature of about 39° C.

TABLE 4 Reassortment analysis of the EIV-P821 39° C. protein synthesisphenotype with avian influenza virus, A/mallard/New York/6750/78 GeneVirus ts+¹ ts−² HA avian 18 1 equine 11 7 NP avian 34 5 equine 7 8 NS-1avian 10 4 equine 14 5¹number of clonal isolates which synthesize all viral proteins at atemperature of about 39° C.²number of clonal isolates inhibited in the ability to synthesize allviral proteins at a temperature of about 39° C.

The results demonstrated an association of the equine NP gene with amutation causing the inability of EIV-P821 to form plaques at anon-permissive temperature of about 39° C., but the results did notsuggest an association of any of the HA, NP, or NS-1 genes with amutation causing the inability of EIV-P821 to express viral proteins ata non-permissive temperature of about 39° C. Thus, these data alsodemonstrated that the plaque formation phenotype and the proteinsynthesis phenotype observed in virus EIV-P821 were the result ofseparate mutations.

D. Studies were also conducted to determine if cold-adapted equineinfluenza viruses of the present invention have a dominant interferencephenotype, that is, whether they dominate in mixed infection with thewild type parental virus A/Kentucky/1/91 (H3N8). The dominantinterference phenotype of viruses EIV-P821 and EIV-P824 were evaluatedin the following manner. Separate monolayers of MDCK cells were singlyinfected with the parental virus A/Kentucky/1/91 (H3N8) at an MOI of 2,singly infected with either cold-adapted virus EIV-P821 or EIV-P824 atan MOI of 2, or simultaneously doubly infected with both the parentalvirus and one of the cold adapted viruses at an MOT of 2+2, all at atemperature of about 34° C. At 24 hours after infection, the media fromthe cultures were harvested and the virus yields from the variousinfected cells were measured by duplicate plaque assays performed attemperatures of about 34° C. and about 39° C. This assay took advantageof the fact that cold adapted equine influenza viruses EIV-P821 orEIV-P824 are temperature sensitive and are thus unable to form plaquesat a non-permissive temperature of about 39° C., while the parentalvirus is able to form plaques at both temperatures, thus making itpossible to measure the growth of the parental virus in the presence ofthe cold adapted virus. Specifically, the dominant interference effectof the cold adapted virus on the growth of the parental virus wasquantitated by comparing the virus yield at about 39° C. of the cellssingly infected with parental virus to the yield of the parental virusin doubly infected cells. EIV-P821, in mixed infection, was able toreduce the yield of the parental virus by approximately 200 fold, whileEIV-P824, in mixed infection, reduced the yield of the parental virus byapproximately 3200 fold. This assay therefore showed that cold-adaptedequine influenza viruses EIV-P821 and EIV-P824 both exhibit the dominantinterference phenotype.

E. Virus isolate EIV-MSV+5 was derived from EIV-P821, as follows.EIV-P821 was passaged once in eggs, as described above, to produce aMaster Seed Virus isolate, denoted herein as EIV-MSV0. EIV-MSV0 was thensubjected to passage three additional times in eggs, the virus isolatesat the end of each passage being designated EIV-MSV+1, EIV-MSV+2, andEIV-MSV+3, respectively. EIV-MSV+3 was then subjected to two additionalpassages in MDCK cells, as follows. MDCK cells were grown in 150 cm²tissue culture flasks in MEM tissue culture medium with Hanks Salts,containing 10% calf serum. The cells were then washed with sterile PBSand the growth medium was replaced with about 8 ml per flask ofinfection medium (tissue culture medium comprising MEM with Hanks Salts,1 μg/ml TPCK trypsin solution, 0.125% bovine serum albumin (BSA), and 10mM HEPES buffer). MDCK cells were inoculated with AF containing virusEIV-MSV+3 (for the first passage in MDCK cells) or virus stock harvestedfrom EIV-MSV+4 (for the second passage in MDCK cells), and the viruseswere allowed to adsorb for 1 hour at about 34° C. The inoculum wasremoved from the cell monolayers, the cells were washed again with PBS,and about 100 ml of infection medium was added per flask. The infectedcells were incubated at about 34° C. for 24 hours. The virus-infectedMDCK cells were harvested by shaking the flasks vigorously to disruptthe cell monolayer, resulting in virus isolates EIV-MSV+4 (the firstpassage in MDCK cells), and EIV-MSV+5 (the second passage in MDCKcells).

Viruses EIV-MSV0 and EIV-MSV+5 were subjected to phenotypic analysis, asdescribed in section B above, to determine their ability to form plaquesand synthesize viral proteins at temperatures of about 34° C., about 37°C., and about 39° C. Both EIV-MSV0 and EIV-MSV+5 formed plaques intissue culture cells at a temperature of about 34° C., and neither virusisolate formed plaques or exhibited detectable viral protein synthesisat a temperature of about 39° C. Virus EIV-MSV0 had a similartemperature sensitive phenotype as EIV-P821 at a temperature of about37° C., i.e., it was inhibited in plaque formation, and late geneexpression was inhibited. However, EIV-MSV+5, unlike its parent virus,EIV-P821, did form plaques in tissue culture at a temperature of about37° C., and at this temperature, the virus synthesized normal amounts ofall proteins. Virus EIV-MSV+5 has been deposited with the ATCC underAccession No. ATCC VR-______.

EXAMPLE 2

Therapeutic compositions of the present invention were produced asfollows.

A. A large stock of ETV-P821 was propagated in eggs as follows. About 60specific pathogen-free embryonated chicken eggs were candled andnon-viable eggs were discarded. Stock virus was diluted to about 1.0×10⁵pfu/ml in sterile PBS. Virus was inoculated into the allantoic cavity ofthe eggs as described in Example 1A. After a 3-day incubation in ahumidified chamber at a temperature of about 34° C., AF was harvestedfrom the eggs according to the method described in Example 1A. Theharvested AF was mixed with a stabilizer solution, for example A1/A2stabilizer, available from Diamond Animal Health, Des Moines, Iowa, at25% V/V (stabilizer/AF). The harvested AU was batched in a centrifugetube and was clarified by centrifugation for 10 minutes at 1000 rpm inan IEC Centra-7R refrigerated table top centrifuge fitted with aswinging bucket rotor. The clarified fluid was distributed into 1-mlcryovials and was frozen at about −70° C. Virus stocks were titrated onMDCK cells by CPE and plaque assay at about 34° C.

B. A large stock of EIV-P821 was propagated in MDCK cells as follows.MDCK cells were grown in 150 cm² tissue culture flasks in MEM tissueculture medium with Hanks Salts, containing 10% calf serum. The cellswere then washed with sterile PBS and the growth medium was replacedwith about 8 ml per flask of infection medium. The MDCK cells wereinoculated with virus stock at an MOI ranging from about 0.5 pfu percell to about 0.005 pfu per cell, and the viruses were allowed to adsorbfor 1 hour at about 34° C. The inoculum was removed from the cellmonolayers, the cells were washed again with PBS, and about 100 ml ofinfection medium was added per flask. The infected cells were incubatedat about 34° C. for 24 hours. The virus-infected MDCK cells wereharvested by shaking the flasks vigorously to disrupt the cell monolayerand stabilizer solution was added to the flasks at 25% V/V(stabilizer/virus solution). The supernatants were distributedaseptically into cryovials and frozen at −70° C.

C. Therapeutic compositions comprising certain cold-adapted temperaturesensitive equine influenza viruses of the present invention wereformulated as follows. Just prior to vaccination procedures, such asthose described in Examples 3-7 below, stock vials of EIV-P821 orEIV-MSV+5 were thawed and were diluted in an excipient comprising eitherwater, PBS, or in MEM tissue culture medium with Hanks Salts, containing0.125% bovine serum albumin (BSA-MEM solution) to the desired dilutionfor administration to animals. The vaccine compositions were held on iceprior to vaccinations. All therapeutic compositions were titered on MDCKcells by standard methods just prior to vaccinations and whereverpossible, an amount of the composition, treated identically to thoseadministered to the animals, was titered after the vaccinations toensure that the virus remained viable during the procedures.

EXAMPLE 3

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821 was tested for safety and its ability to replicate in threehorses showing detectable prior immunity to equine influenza virus asfollows. EIV-P821, produced as described in Example 1A, was grown ineggs as described in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu EIV-P821/2 ml BSA-MEM solution asdescribed in Example 2C.

Three ponies having prior detectable hemagglutination inhibition (HAI)titers to equine influenza virus were inoculated with a therapeuticcomposition comprising EIV-P821 by the following method. Each pony wasgiven a 2-ml dose of EIV-P821, administered intranasally using a syringefitted with a blunt cannula long enough to reach past the false nostril,1 ml per nostril.

The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination forimmediate type allergic reactions such as sneezing, salivation, laboredor irregular breathing, shaking, anaphylaxis, or fever. The animals werefurther monitored on days 1-11 post-vaccination for delayed typeallergic reactions, such as lethargy or anorexia. None of the threeponies in this study exhibited any allergic reactions from thevaccination.

The ponies were observed daily, at approximately the same time each day,starting two days before vaccination and continuing through day 11following vaccination for clinical signs consistent with equineinfluenza. The ponies were observed for nasal discharge, oculardischarge, anorexia, disposition, heart rate, capillary refill time,respiratory rate, dyspnea, coughing, lung sounds, presence of toxic lineon upper gum, and body temperature. In addition submandibular andparietal lymph nodes were palpated and any abnormalities were described.None of the three ponies in this study exhibited any abnormal reactionsor overt clinical signs during the observation period.

To test for viral shedding in the animals, on days 0 through 11following vaccination, nasopharyngeal swabs were collected from theponies as described in Chambers, et al., 1995, Equine Practice, 17,19-23. Chambers, et al., ibid. Briefly, two sterile Dacron polyestertipped applicators (available, e.g., from Hardwood Products Co.,Guilford, Me.) were inserted, together, into each nostril of the ponies.The swabs (four total, two for each nostril) were broken off into a15-ml conical centrifuge tube containing 2.5 ml of chilled transportmedium comprising 5% glycerol, penicillin, streptomycin, neomycin, andgentamycin in PBS at physiological pH. Keeping the samples on wet ice,the swabs were aseptically wrung out into the medium and thenasopharyngeal samples were divided into two aliquots. One aliquot wasused to attempt isolation of EIV by inoculation of embryonated eggs,using the method described in Example 1. The AF of the inoculated eggswas then tested for its ability to cause hemagglutination, by standardmethods, indicating the presence of equine influenza virus in the AF. Ondays 2 and 3 post-vaccination, the other aliquots were tested for virusby the Directigen® Flu A test, available from Becton-Dickinson(Cockeysville, Md.).

Attempts to isolate EIV from the nasopharyngeal secretions of the threeanimals by egg inoculation were unsuccessful. However on days 2 and 3,all animals tested positive for the presence of virus shedding using theDirectigen Flu A test, consistent with the hypothesis that EIV-P821 wasreplicating in the seropositive ponies.

To test the antibody titers to EIV in the inoculated animals describedin this example, as well as in the animals described in Examples 4-7,blood was collected from the animals prior to vaccination and ondesignated days post-vaccination. Serum was isolated and was treatedeither with trypsin/periodate or kaolin to block the nonspecificinhibitors of hemagglutination present in normal sera. Serum sampleswere tested for hemagglutination inhibition (HAI) titers against arecent EIV isolate by standard methods, described, for example in the“Supplemental assay method for conducting the hemagglutinationinhibition assay for equine influenza virus antibody” (SAM 124),provided by the U.S.D.A. National Veterinary Services Laboratory under 9CFR 113.2.

The HAI titers of the three ponies are shown in Table 5. As can be seen,regardless of the initial titer, the serum HAI titers increased at leastfour-fold in all three animals after vaccination with EIV-P821.

These data demonstrate that cold-adapted equine influenza virus EIV-P821is safe and non-reactogenic in sero-positive ponies, and that theseanimals exhibited an increase in antibody titer to equine influenzavirus, even though they had prior demonstrable titers. TABLE 5 HAItiters of vaccinated animals* Animal HAI Titer (days after vaccination)ID 0 7 14 21 18 40 80 160 160 19 10 20 40 80 25 20 40 320 80*HAI titers are expressed as the reciprocal of the highest dilution ofserum which inhibited hemagglutination of erythrocytes by a recentisolate of equine influenza virus.

EXAMPLE 4

This Example discloses an animal study to evaluate the safety andefficacy of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821.

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821 was tested for attenuation, as well as its ability to protecthorses from challenge with virulent equine influenza virus, as follows.EIV-P821, produced as described in Example 1, was grown in eggs asdescribed in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu of virus/2 ml water, as described inExample 2C. Eight EIV-seronegative ponies were used in this study. Threeof the eight ponies were vaccinated with a 2-ml dose comprising 10⁷ pfuof the EIV-P821 therapeutic composition, administered intranasally,using methods similar to those described in Example 3. One pony wasgiven 10⁷ pfu of the EIV-P821 therapeutic composition, administeredorally, by injecting 6 ml of virus into the pharynx, using a 10-mlsyringe which was adapted to create a fine spray by the followingmethod. The protruding “seat” for the attachment of needles was sealedoff using modeling clay and its cap was left in place. About 10 holeswere punched through the bottom of the syringe, i.e., surrounding the“seat,” using a 25-gauge needle. The syringe was placed into theinterdental space and the virus was forcefully injected into the back ofthe mouth. The remaining four ponies were held as non-vaccinatedcontrols.

The vaccinated ponies were observed for approximately 30 minutesimmediately following and at approximately four hours after vaccinationfor immediate type allergic reactions, and the animals were furthermonitored on days 1-11 post-vaccination for delayed type allergicreactions, both as described in Example 3. None of the four vaccinatedponies in this study exhibited any abnormal reactions from thevaccination.

The ponies were observed daily, at approximately the same time each day,starting two days before virus vaccination and continuing through day 11following vaccination for clinical signs, such as those described inExample 3. None of the four vaccinated ponies in this study exhibitedany clinical signs during the observation period. This resultdemonstrated that cold-adapted equine influenza virus EIV-P821 exhibitsthe phenotype of attenuation.

To test for viral shedding in the vaccinated animals, on days 0 through11 following vaccination, nasopharyngeal swabs were collected from theponies as described in Example 3. The nasopharyngeal samples were testedfor virus in embryonated chicken eggs according to the method describedin Example 3.

As shown in Table 6, virus was isolated from only one vaccinated animalusing the egg method. However, as noted in Example 3, the lack ofisolation by this method does not preclude the fact that virusreplication is taking place, since replication may be detected by moresensitive methods, e.g., the Directigen Flu A test. TABLE 6 Virusisolation in eggs after vaccination. Animal Virus Isolation (days aftervaccination) ID Route 0 1 2 3 4 5 6 7 8 9 10 11 91 IN −− + + + + + + + + + − 666 IN − − − − − − − − − − − − 673 IN − − − − − −− − − − − − 674 Oral − − − − − − − − − − − −

To test the antibody titers to equine influenza virus in the vaccinatedanimals, blood was collected from the animals prior to vaccination andon days 7, 14, 21, and 28 post-vaccination. Serum samples were isolatedand were tested for hemagglutination inhibition (HAT) titers against arecent EIV isolate according to the methods described in Example 3.

The HAI titers of the four vaccinated ponies are shown in Table 7. TABLE7 HAI titers after vaccination. Animal HAI Titer (days aftervaccination) ID Route 0 7 14 21 28 91 IN <10 <10 <10 <10 <10 666 IN 1010 10 20 20 673 IN 10 10 10 20 20 674 Oral 20 40 40 40 40

Unlike the increase in HAI titer observed with the three animalsdescribed in the study in Example 3, the animals in this study did notexhibit a significant increase, i.e., greater than four-fold, in HAItiter following vaccination with EIV-P821.

Approximately four and one-half months after vaccine virusadministration, all 8 ponies, i.e., the four that were vaccinated andthe four non-vaccinated controls, were challenged by the followingmethod. For each animal, 10⁷ pfu of the virulent equine influenza virusstrain A/equine/Kentucky/1/91 (H3N8) was suspended in 5 ml of water. Amask was connected to a nebulizer, and the mask was placed over theanimal's muzzle, including the nostrils. Five (5) ml was nebulized foreach animal, using settings such that it took 5-10 minutes to deliverthe full 5 ml. Clinical observations, as described in Example 3, wereperformed on all animals three days before challenge and daily for 11days after challenge.

Despite the fact that the vaccinated animals did not exhibit markedincreases in their HAI titers to equine influenza virus, all fourvaccinated animals were protected against equine influenza viruschallenge. None of the vaccinated animals showed overt clinical signs orfever, although one of the animals had a minor wheeze for two days. Onthe other hand, all four non-vaccinated ponies shed virus and developedclinical signs and fever typical of equine influenza virus infection.Thus, this example demonstrates that a therapeutic composition of thepresent invention can protect horses from equine influenza disease.

EXAMPLE 5

This Example discloses an additional animal study to evaluateattenuation of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821, and its ability to protect vaccinated horsesfrom subsequent challenge with virulent equine influenza virus.Furthermore, this study evaluated the effect of exercise stress on thesafety and efficacy of the therapeutic composition.

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821 was tested for safety and efficacy in horses, as follows.EIV-P821, produced as described in Example 1, was grown in eggs asdescribed in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu virus/5 ml water, as described in Example2C. Fifteen ponies were used in this study. The ponies were randomlyassigned to three groups of five animals each, as shown in Table 8,there being two vaccinated groups and one unvaccinated control group.The ponies in group 2 were exercise stressed before vaccination, whilethe ponies in vaccinate group 1 were held in a stall. TABLE 8Vaccination/challenge protocol. Group No. Ponies Exercise VaccineChallenge 1 5 — Day 0 Day 90 2 5 Days −4 to 0 Day 0 Day 90 3 5 — — Day90

The ponies in group 2 were subjected to exercise stress on a treadmillprior to vaccination, as follows. The ponies were acclimated to the useof the treadmill by 6 hours of treadmill use at a walk only. The actualexercise stress involved a daily exercise regimen starting 4 days beforeand ending on the day of vaccination (immediately prior to vaccination).The treadmill exercise regimen is shown in Table 9. TABLE 9 Exerciseregimen for the ponies in Group 2. Speed (m/sec) Time (min.) Incline (°)1.5 2 0 3.5 2 0 3.5 2 7  4.5 † 2 7  5.5 † 2 7  6.5 † 2 7  7.5 † 2 7  8.5† 2 7 3.5 2 7 1.5 10  0 ††Speed, in meters per second (m/sec) was increased for each animal every2 minutes until the heart rate reached and maintained ≧200 beats perminute

Groups 1 and 2 were given a therapeutic composition comprising 10⁷ pfuof EIV-P821, by the nebulization method described for the challengedescribed in Example 4. None of the vaccinated ponies in this studyexhibited any immediate or delayed allergic reactions from thevaccination.

The ponies were observed daily, at approximately the same time each day,starting two days before vaccination and continuing through day 11following vaccination for clinical signs, such as those described inExample 3. None of the vaccinated ponies in this study exhibited anyovert clinical signs during the observation period.

To test for viral shedding in the vaccinated animals, before vaccinationand on days 1 through 11 following vaccination, nasopharyngeal swabswere collected from the ponies as described in Example 3. Thenasopharyngeal samples were tested for virus in embryonated chicken eggsaccording to the method described in Example 3. Virus was isolated fromthe vaccinated animals, i.e., Groups 1 and 2, as shown in Table 10.TABLE 10 Virus isolation after vaccination. Animal Virus Isolation (daysafter vaccination) Group ID Exercise 0 1 2 3 4 5 6 7 8 9 10 11 1 12 No −− + + + + + − + + − − 16 − − + + + + + − − − − − 17 − − + + + + + + +− + − 165 − − − − − − − − − − − − 688 − − − − − + − + − − − − 2 7 Yes −− − + + + + − − − − − 44 − − − − − − − − − − − − 435 − − + + + + − − − −− − 907 − − − + − + + − − − − − 968 − − − − − + − + − − − −

To test the antibody titers to equine influenza virus in the vaccinatedanimals, blood was collected prior to vaccination and on days 7, 14, 21,and 28 post-vaccination. Serum samples were isolated and were tested forHAI titers against a recent EIV isolate according to the methodsdescribed in Example 3. These titers are shown in Table 11. TABLE 11 HAItiters after vaccination and after challenge on day 90. Animal DayPost-vaccination Group ID −1 7 14 21 28 91 105 112 119 126 1 12 <10 <10<10 <10 <10 <10 80 320 320 640 1 16 <10 <10 20 20 <10 <10 20 160 320 3201 17 <10 <10 10 10 10 10 80 160 160 160 1 165 <10 <10 10 10 10 10 80 8080 80 1 688 <10 <10 20 20 20 20 20 20 20 40 2 7 <10 <10 10 10 <10 <10 2080 80 40 2 44 <10 <10 20 20 20 10 80 320 320 320 2 435 <10 <10 20 20 10<10 20 80 80 80 2 907 <10 <10 10 10 20 10 10 40 80 80 2 968 <10 <10 <10<10 <10 <10 40 160 160 160 3 2 <10 80 640 640 320 3 56 <10 80 320 320320 3 196 <10 20 160 80 80 3 198 10 40 160 320 320 3 200 <10 20 80 80 40Group Description 1 Vaccination only 2 Vaccination and Exercise 3Control

On day 90 post vaccination, all 15 ponies were challenged with 10⁷ pfuof equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by thenebulizer method as described in Example 4. Clinical observations, asdescribed in Example 3, were performed on all animals three days beforechallenge and daily for 11 days after challenge. There were no overtclinical signs observed in any of the vaccinated ponies. Four of thefive non-vaccinated ponies developed fever and clinical signs typical ofequine influenza virus infection.

Thus, this example demonstrates that a therapeutic composition of thepresent invention protects horses against equine influenza disease, evenif the animals are stressed prior to vaccination.

EXAMPLE 6

This Example compared the infectivities of therapeutic compositions ofthe present invention grown in eggs and grown in tissue culture cells.From a production standpoint, there is an advantage to growingtherapeutic compositions of the present invention in tissue culturerather than in embryonated chicken eggs. Equine influenza virus,however, does not grow to as high a titer in cells as in eggs. Inaddition, the hemagglutinin of the virus requires an extracellularproteolytic cleavage by trypsin-like proteases for infectivity. Sinceserum contains trypsin inhibitors, virus grown in cell culture must bepropagated in serum-free medium that contains trypsin in order to beinfectious. It is well known by those skilled in the art that suchconditions are less than optimal for the viability of tissue culturecells. In addition, these growth conditions may select for virus withaltered binding affinity for equine cells, which may affect viralinfectivity since the virus needs to bind efficiently to the animal'snasal mucosa to replicate and to stimulate immunity. Thus, the objectiveof the study disclosed in this example was to evaluate whether theinfectivity of therapeutic compositions of the present invention wasadversely affected by growth for multiple passages in in vitro tissueculture.

EIV-P821, produced as described in Example 1, was grown in eggs asdescribed in Example 2A or in MDCK cells as described in Example 2B. Ineach instance, the virus was passaged five times. EIV-P821 was testedfor its cold-adaptation and temperature sensitive phenotypes after eachpassage. The egg and cell-passaged virus preparations were formulatedinto therapeutic compositions comprising 10⁷ pfu virus/2 ml BSA-MEMsolution, as described in Example 2C, resulting in an egg-grown EIV-P821therapeutic composition and an MDCK cell-grown EIV-P821 therapeuticcomposition, respectively.

Eight ponies were used in this study. Serum from each of the animals wastested for HAI titers to equine influenza virus prior to the study. Theanimals were randomly assigned into one of two groups of four ponieseach. Group A received the egg-grown EIV-P821 therapeutic composition,and Group B received the MDCK-grown EIV-P821 therapeutic composition,prepared as described in Example 2B. The therapeutic compositions wereadministered intranasally by the method described in Example 3.

The ponies were observed daily, at approximately the same time each day,starting two days before vaccination and continuing through day 11following vaccination for allergic reactions or clinical signs asdescribed in Example 3. No allergic reactions or overt clinical signswere observed in any of the animals.

Nasopharyngeal swabs were collected before vaccination and daily for 11days after vaccination. The presence of virus material in the nasalswabs was determined by the detection of CPE on MDCK cells infected asdescribed in Example 1, or by inoculation into eggs and examination ofthe ability of the infected AF to cause hemagglutination, as describedin Example 3. The material was tested for the presence of virus only,and not for titer of virus in the sample. Virus isolation results arelisted in Table 12. Blood was collected and serum samples from days 0,7, 14, 21 and 28 after vaccination were tested for hemagglutinationinhibition antibody titer against a recent isolate. HAI titers are alsolisted in Table 12. TABLE 12 HAI titers and virus isolation aftervaccination. HAI Titer (DPV³) Virus Isolation ¹ (DPV³) Group² ID 0 7 1421 28 0 1 2 3 4 5 6 7 8 9 10 11 1 31 <10 20 160 160 160 — EC — C EC EC CC EC — — — 37 <10 40 160 160 160 — EC C C EC C C C — — — — 40 <10 20 80160 80 — EC EC C — C EC C — EC EC — 41 <10 40 160 160 80 — EC EC C EC CEC EC — — — — 2 32 <10 <10 80 80 40 — EC — C — C — C — EC — — 34 <10 20160 160 160 — EC — C EC C EC C — — — — 35 <10 <10 80 80 40 — EC — C — C— C — EC — — 42 <10 <10 80 80 40 — — — C — C EC EC — — — —¹E = Egg isolation positive; C = CPE isolation positive; — = virus notdetected by either of the methods²Group 1: Virus passaged 5× in MDCK cells; Group 2: Virus passaged 5× inEggs³Days Post-vaccination

The results in Table 12 show that there were no significant differencesin infectivity or immunogenicity between the egg-grown and MDCK-grownEIV-P821 therapeutic compositions.

EXAMPLE 7

This example evaluated the minimum dose of a therapeutic compositioncomprising a cold-adapted equine influenza virus required to protect ahorse from equine influenza virus infection.

The animal studies disclosed in Examples 3-6 indicated that atherapeutic composition of the present invention was efficacious andsafe. In those studies, a dose of 10⁷ pfu, which correlates toapproximately 10⁸ TCID₅₀units, was used. However, from the standpointsof cost and safety, it is advantageous to use the minimum virus titerthat will protect a horse from disease caused by equine influenza virus.In this study, ponies were vaccinated with four different doses of atherapeutic composition comprising a cold-adapted equine influenza virusto determine the minimum dose which protects a horse against virulentequine influenza virus challenge.

EYV-P821, produced as described in Example 1A, was passaged and grown inMDCK cells as described in Example 2B and was formulated into atherapeutic composition comprising either 2×10⁴, 2×10⁵, 2×10⁶, or 2×10⁷TCID₅₀, units/1 ml BSA-MEM solution as described in Example 2C. Nineteenhorses of various ages and breeds were used for this study. The horseswere assigned to four vaccine groups, one group of three horses andthree groups of four horses, and one control group of four horses (seeTable 13). Each of the ponies in the vaccine groups were given a 1-mldose of the indicated therapeutic composition, administered intranasallyby methods similar to those described in Example 3. TABLE 13 Vaccinationprotocol. Group No. No. Animals Vaccine Dose, TCID₅₀ Units 1 3 2 × 10⁷ 24 2 × 10⁶ 3 4 2 × 10⁵ 4 4 2 × 10⁴ 5 4 control

The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination forimmediate type reactions, and the animals were further monitored on days1-11 post-vaccination for delayed type reactions, both as described inExample 3. None of the vaccinated ponies in this study exhibited anyabnormal reactions or overt clinical signs from the vaccination.

Blood for serum analysis was collected 3 days before vaccination, ondays 7, 14, 21, and 28 after vaccination, and after challenge on Days 35and 42. Serum samples were tested for HAI titers against a recent EIVisolate according to the methods described in Example 3. These titersare shown in Table 14. Prior to challenge on day 29, 2 of the 3 animalsin group 1, 4 of the 4 animals in group 2, 3 of the 4 animals in group3, and 2 of the 4 animals in group 4 showed at least 4-fold increases inHAI titers after vaccination. In addition, 2 of the 4 control horsesalso exhibited increases in HAI titers. One interpretation for thisresult is that the control horses were exposed to vaccine virustransmitted from the vaccinated horses, since all the horses in thisstudy were housed in the same barn. TABLE 14 HAI titers post-vaccinationand post-challenge, and challenge results. Dose in Animal Vaccination onDay 0, Challenge on Day 29 Chall. Sick No. TCID₅₀ units ID −1 7 14 21 2835 42 +/− 1 2 × 10⁷ 41 <10 <10 10 40 10 20 80 − 42 40 40 40 40 40 <10 80− 200 <10 <10 80 40 160 40 40 − 2 2 × 10⁶ 679 <10 10 40 40 40 20 20 −682 <10 <10 40 40 40 40 40 − 795 20 80 160 160 320 320 640 − R <10 10 4020 160 40 40 − 3 2 × 10⁵ 73 <10 <10 160 40 80 160 160 − 712 <10 <10 2020 40 40 20 − 720 <10 20 80 40 80 80 160 − 796 <10 <10 <10 <10 <10 1080 + 4 2 × 10⁴ 75 <10 <10 <10 <10 <10 <10 160 + 724 <10 >10 <10 <10 <1020 320 + 789 <10 10 320 160 320 320 320 − 790 <10 <10 80 40 160 80 40 5Control 12 <10 <10 <10 20 20 40 40 − 22 10 20 40 10 160 40 640 − 71 <10<10 <10 <10 10 20 160 + 74 <10 <10 <10 <10 <10 <10 20 +

On day 29 post vaccination, all 19 ponies were challenged with equineinfluenza virus strain a/equine/Kentucky/1/91 (H3N8) by the nebulizermethod as described in Example 4. The challenge dose was prospectivelycalculated to contain about 10⁸ TCID₅₀ units of challenge virus in avolume of 5 ml for each animal. Clinical observations, as described inExample 3, were monitored beginning two days before challenge, the dayof challenge, and for 11 days following challenge. As shown in Table 14,no animals in groups 1 or 2 exhibited clinical signs indicative ofequine influenza disease, and only one out of four animals in group 3became sick. Two out of four animals in group 4 became sick, and onlytwo of the four control animals became sick. The results in Table 14suggest a correlation between seroconversion and protection fromdisease, since, for example, the two control animals showing increasedHAI titers during the vaccination period did not show clinical signs ofequine influenza disease following challenge. Another interpretation,however, was that the actual titer of the challenge virus may have beenless than the calculated amount of 10⁸ TCID₅₀ units, since, based onprior results, this level of challenge should have caused disease in allthe control animals.

Nonetheless, the levels of seroconversion and the lack of clinical signsin the groups that received a therapeutic composition comprising atleast 2×10⁶ TCID₅₀ units of a cold-adapted equine influenza virussuggests that this amount was sufficient to protect a horse againstequine influenza disease. Furthermore, a dose of 2×10⁵ TCID₅₀ unitsinduced seroconversion and gave clinical protection from challenge in 3out of 4 horses, and thus even this amount may be sufficient to confersignificant protection in horses against equine influenza disease.

EXAMPLE 8

This example discloses an animal study to evaluate the duration ofimmunity of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821.

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821, produced as described in Example 1, was grown in eggssimilarly to the procedure described in Example 2A, was expanded bypassage in MDCK cells similarly to the procedure described in Example2B, and was formulated into a therapeutic composition as described inExample 2C. Thirty horses approximately 11 to 12 months of age were usedfor this study. Nineteen of the horses were each vaccinated intranasallyinto one nostril using a syringe with a delivery device tip attached tothe end, with a 1.0 ml dose comprising 6 logs of TCID₅₀ units of theEIV-P821 therapeutic composition. Vaccinations were performed on Day 0.

The horses were observed on Day 0 (before vaccination and up to 4 hourspost-vaccination) and on Study Days 1, 2, 3, 7, 15, and 169post-vaccination. On these days, a distant examination for a period ofat least 15 minutes was performed. This distant examination includedobservation for demeanor, behavior, coughing, sneezing, and nasaldischarge. The examination on Day 169 also served to confirm that thehorses were in a condition of health suitable for transport to thechallenge site which was located approximately 360 miles from thevaccination site.

The animals were acclimated to the challenge site and were observedapproximately daily by a veterinarian or animal technician for evidenceof disease. A general physical examination was performed on Day 171post-vaccination to monitor the following: demeanor, behavior, coughing,sneezing, and nasal discharge. From Days 172 to 177, similarobservations as well as rectal temperature were recorded, according tothe judgment of the attending veterinarian for any individual horse withabnormal clinical presentation.

No vaccinated horses showed any adverse reactions post-vaccination. Onevaccinate was found dead about two months after vaccination. This horseshowed no evidence of adverse reaction when observed for at least onemonth after vaccination. Although no cause of death could be firmlyestablished, the death was not instantaneous and was considered to beconsistent with possible contributing factors such as colic, bonefracture, or severe worm burden. Since there was no other evidence forany adverse reactions post-vaccination in any other vaccinates, it ishighly unlikely that the vaccine contributed to any adverse reaction inthis case.

Challenges were performed on Day 181 post-vaccination. The followingwild-type isolate of equine influenza virus previously shown to causedisease in horses was used as the challenge virus:A/equine/2/Kentucky/91. Prior to infection of each challenge group, thechallenge material was rapidly thawed at approximately 37° C. The viruswas diluted with phosphate-buffered saline to a total volume ofapproximately 21 ml. The diluted material was stored chilled on iceuntil immediately before inoculation. Before inoculation and at the endof nebulization for each challenge group, a sample of diluted challengevirus was collected for pre-and post-inoculation virus titerconfirmation. Vaccinates and controls were randomly assigned to 4challenge groups of 6 horses each and one challenge group of 5 horses sothat each challenge group contained a mixture of 4 vaccinates and 2controls or 3 vaccinates and 2 controls.

Challenge virus in aerosol form was delivered through a tube insertedthrough a small opening centrally in the plastic ceiling with anultrasonic nebulizer (e.g., DeVilbiss Model 099HD, DeVilbiss HealthcareInc., Somerset, Pa.) for a period of approximately 10 minutes. Thehorses remained in the chamber for a further period of approximately 30minutes after the nebulization had been completed (total exposure time,approximately 40 minutes). At that time, the plastic was removed to ventthe chamber, and the horses were released and returned to their pen. Thechallenge procedure was repeated for each group.

All statistical methods in this study were performed using SAS (SASInstitute, Cary, N.C.), and P<0.05 was considered to be statisticallysignificant. Beginning on Day 178 post-vaccination (three days prior tochallenge) through Day 191 (day 10 post-challenge), the horses wereobserved daily by both distant and individual examinations. Rectaltemperatures were measured at these times. Data from day 0 (challengeday) to day 10 were included in the analysis; see Table 15. TABLE 15Effect of challenge on daily temperatures (° C.) in vaccinated andcontrol horses (least squares means). Vaccinated non-vaccinated Day postchallenge (n = 19) (n = 10) P-value 0 100.7 100.8 0.8434 1 100.5 100.40.7934 2 103.4 104.9 0.0024 3 101.8 103.9 0.0001 4 101.5 103.2 0.0002 5101.7 103.8 0.0001 6 101.3 103.6 0.0001 7 100.7 102.3 0.0007 8 100.5101.4 0.0379 9 100.1 100.3 0.7416 10 100.3 100.5 0.7416 pooled SEM* 0.270.38*Standard error of the meanTable 15 shows that on days 2 through 8, vaccinated horses had lowertemperatures (P<0.05) than the non-vaccinated control horses.

The distant examination consisted of a period of 20 minutes where thefollowing observations were made: coughing, nasal discharge,respiration, and depression. Scoring criteria are shown in Table 16.TABLE 16 Clinical signs and scoring index. Clinical Sign DescriptionScore Coughing normal during observation period of 15 min 0 coughingonce during observation 1 coughing twice or more during observation 2Nasal discharge normal 0 abnormal, serous 1 abnormal, mucopurulent 2abnormal, profuse 3 Respiration normal 0 abnormal (dyspnea, tachypnea) 1Depression normal 0 depression present^(†) 1^(†)Depression was assessed by subjective evaluation of individualanimal behavior that included the following: failure to approach foodrapidly, general lethargy, inappetence, and anorexia.

Each horse was scored for each of these categories. Additionally,submandibular lymph nodes were palpated to monitor for possiblebacterial infection. In any case where there was a different valuerecorded for a subjective clinical sign score from an observation on thesame day at the distant versus the individual examination, the greaterscore was used in the compilation and analysis of results. For purposesof assessing the health of the horses prior to final disposition,distant examinations were performed at 14, 18, and 21 dayspost-challenge. Data from days 1 through 10 post-challenge were includedin the analysis. These scores were summed on each day for each horse,and the vaccinates and controls were compared using the Wilcoxon ranksums test. In addition, these scores were summed across all days foreach horse, and compared in the same manner. The mean ranks and meanclinical scores are shown in Tables 17 and 18, respectively. Five dayspost-challenge, the mean rank of scores in the vaccinated horses waslower (P<0.05) than in the non-vaccinated control horses; and thiseffect continued on days 6, 7, 8, 9, and 10 (P<0.05). The cumulativerank over the entire test period was also lower (P<0.05) in thevaccinated horses than the non-vaccinated controls. TABLE 17 Effect ofchallenge on clinical sign scores in vaccinated and control horses (meanrank). Vaccinated Non-vaccinated (n = 19), (n = 10), Day post challengemean rank* mean rank P-value 0 13.6 17.6 0.1853 1 16.4 12.4 0.2015 215.1 14.9 0.9812 3 13.3 18.3 0.1331 4 13.5 17.9 0.1721 5 12.4 19.90.0237 6 12.7 19.4 0.0425 7 12.1 20.6 0.0074 8 12.6 19.6 0.0312 9 13.118.7 0.0729 10 12.3 20.1 0.0135 total over 11 days 11.8 21.2 0.0051*By Wilcoxon rank sum test.

TABLE 18 Effect of challenge on clinical sign scores in vaccinated andcontrol horses (mean scores). Day post challenge Vaccinated (n = 19)Non-vaccinated (n = 10) 0 1.2 1.6 1 1.5 0.9 2 2.4 2.5 3 3.2 4.1 4 3.44.3 5 3.2 4.7 6 3.4 4.8 7 3.3 4.7 8 3.2 4.5 9 3.2 3.9 10 2.4 3.4

Nasopharyngeal swabs were obtained on the day prior to challenge and ondays 1 to 8 post-challenge, as described in Example 3, and tested forshed virus by cell culture assay. The percent of horses sheddingchallenge virus in each group is shown in Table 19. The percent ofhorses shedding the challenge virus in the vaccinated group was lower(P<0.05) on days 5 and 6 post-challenge than in the non-vaccinatedcontrols. The mean number of days the challenge virus was shed was alsolower (P<0.05) in the vaccinated group as compared to the non-vaccinatedcontrols. TABLE 19 Percent of horses shedding virus per daypost-challenge and mean number of days of shedding per group. Day postchallenge Vaccinated (n = 19) Non-vaccinated (n = 10) −1 0 0 1 63.2 90 2100 100 3 84.2 100 4 100 100 5 47.4 88.9* 6 10.5 77.8* 7 5.3 20 8 0 0average number 4.1 5.6* of days shedding*Within a time point, vaccinates different from non-vaccinates (P <0.05) by either Fisher's exact test (percent data) or Wilcoxon rank sumstest (days shedding).

The scores from clinical signs relevant to influenza and the objectivetemperature measurements both demonstrated a statistically significantreduction in the group of vaccinates when compared to those from thecontrol group; this is consistent with an interpretation that thevaccine conferred significant protection from disease.

The ability of horses to shed influenza virus post-challenge was alsosignificantly reduced in vaccinates as compared to controls in both theincidence of horses positive for shedding on certain days post-challengeand the mean number of days of shedding per horse. This decreasedshedding by vaccinates is important in that it should serve to reducethe potential for exposure of susceptible animals to the wild-type virusin an outbreak of influenza.

The results of this study are consistent with the interpretation thatthe vaccine safely conferred protection for 6 months from clinicaldisease caused by equine influenza and reduced the potential for thespread of naturally occurring virulent equine influenza virus. While thedegree of protection from disease was not complete (13 out of 19vaccinates were protected, while 10/10 controls were sick), there was aclear reduction in the severity and duration of clinical illness and anoticeable effect on the potential for viral shedding after exposure toa virulent strain of equine influenza. The finding that both vaccinatesand controls were seronegative immediately prior to challenge at 6months post-immunization suggests that immunity mediated by somethingother than serum antibody may be of primary importance in the ability ofthis vaccine to confer measurable and durable protection.

EXAMPLE 9

This Example discloses an animal study to evaluate the ability of atherapeutic composition comprising cold-adapted equine influenza virusEIV-P821 to aid in the prevention of disease following exposure to aheterologous strain of equine influenza virus.

The heterologous strain tested was A/equine/2/Saskatoon/90, describedgenetically as a Eurasian strain (obtained from Hugh Townsend,University of Saskatchewan). Twenty female Percheron horsesapproximately 15 months of age (at the time of vaccination) were usedfor the efficacy study. The horses were assigned to two groups, onegroup of 10 to be vaccinated and another group of 10 to serve asnon-vaccinated controls. On day 0, the vaccinate group was vaccinated inthe manner described in Example 8.

The challenge material, i.e. equine flu strain A/equine/2/Saskatoon/90[H3N8] was prepared similarly to the preparation in Example 8.Vaccinates and controls were randomly assigned to 4 challenge groups of5 horses each such that each challenge group contained a mixture of 2vaccinates and three controls or vice versa. The challenge procedure wassimilar to that described in Example 8. Challenges were performed on Day28 post-vaccination.

Clinical observations were performed for the vaccinates and controls onDay −4 and on Study Days 0 (before vaccination and up to 4 hourspost-vaccination), 1 to 7, 12, 15 to 17, 19 to 23, 25 to 38, and 42. Fordays on which clinical observations were performed during Days −4 to 42,clinical observations including rectal temperature were recordedaccording to the judgment of the attending veterinarian for anyindividual horse with abnormal clinical presentation. Horses were scoredusing the same criteria as in Example 8 (Table 15). Distant examinationswere performed on these days as described in Example 8. On Day 20 andfrom Days 25 to 38, the horses were also observed by both distant andindividual examinations (also performed as described in Example 8).

Rectal temperatures were measured daily beginning 3 days prior tochallenge, and continuing until 10 days post-challenge. Day 0 is the dayrelative to challenge. Data from days 0 through 10 were included in theanalysis. Statistical methods and criteria were identical to those usedin Example 8. On days 2, 5 and 7, vaccinated horses had statisticallysignificant lower body temperatures than the non-vaccinated controlhorses (Table 20). TABLE 20 Effect of challenge on daily temperatures (°C.) in vaccinated and control horses (least squares means). VaccinatedNon-vaccinated Day post challenge (n = 10) (n = 10) P-value 0 99.9 99.80.9098 1 100.5 100.3 0.4282 2 101.0 102.8 0.0001 3 100.7 100.6 0.7554 4101.0 101.3 0.4119 5 100.8 102.1 0.0004 6 100.4 100.4 0.9774 7 100.3101.1 0.0325 8 100.6 100.7 0.8651 9 100.5 100.6 0.8874 10 100.5 100.10.2465Standard error of the mean = 0.249.

Data from days 1 through 10 post-challenge were included in theanalysis. These scores were summed on each day for each horse, and thevaccinates and controls were compared using the Wilcoxon rank sums test.All statistical methods were performed as described in Example 9. Inaddition, these scores were summed across all days for each horse, andcompared in the same manner. Mean ranks are shown in Table 21. TABLE 21Effect of challenge on clinical sign scores in vaccinated and controlhorses (mean rank). Vaccinated Non-vaccinated Day post challenge (n =10) (n = 10) P-value* 1 8.85 12.15 0.1741 2 8.80 12.20 0.1932 3 8.9012.10 0.2027 4 7.60 13.40 0.0225 5 6.90 14.10 0.0053 6 7.00 14.00 0.00597 6.90 14.10 0.0053 8 7.60 13.40 0.0251 9 6.90 14.10 0.0048 10 6.1014.90 0.0006 total over 10 days 5.70 15.30 0.0003*By Wilcoxon 2 sample test.

On day 4 post-challenge, the mean rank of scores in the vaccinatedhorses was lower (P<0.05) than the non-vaccinated control horses, andthis effect continued throughout the remainder of the study (P<0.05).The cumulative rank over the entire test period was also lower in thevaccinated horses than the non-vaccinated controls (P<0.05).

Nasopharyngeal swabs were collected on days 1 and 8 post-challenge, asdescribed in Example 3. The nasal samples were analyzed for the presenceof virus by cell inoculation with virus detection by cytopathogeniceffect (CPE) or by egg inoculation with virus detection byhemagglutination (HA). The cell-culture assay was performed as generallydescribed by Youngner et al., 1994, J. Clin. Microbiol. 32, 750-754.Serially diluted nasal samples were added to wells containing monolayersof Madin Darby Canine Kidney (MDCK) cells. After incubation, wells wereexamined for the presence and degree of cytopathogenic effect. Thequantity of virus in TCID₅₀ units was calculated by the Reed-Muenchtechnique. The egg infectivity assay was performed as described inExample 1. The percent of horses shedding challenge virus for each assayin each group is shown in Tables 22 and 23. The percent of horsesshedding the challenge virus in the vaccinated group was lower (P<0.05)on days 2 through 7 post-challenge by either method. No differences wereseen on days 1 or 8 post-challenge. The number of days the challengevirus was shed was also lower (P<0.05) in the vaccinated group ascompared to the non-vaccinated controls; see Tables 22 and 23. TABLE 22Percent of horses shedding virus following challenge - cell cultureassay. Day post challenge Vaccinated (n = 10) Non-vaccinated (n = 10) 10 0 2 0 70* 3 0 70* 4 20 100*  5 10 100*  6 20 100*  7 0 80* 8 0 30 average number 0.5   5.5* of days shedding*Within a time point, vaccinates different from non-vaccinates, P < 0.05by either Fisher's exact test (percent data) or Wilcoxon 2 sample test(days shedding)

TABLE 23 Percent of horses shedding virus following challenge - egginfectivity assay. Day post challenge Vaccinated (n = 10) Non-vaccinated(n = 10) 1 0 0 2 0 70* 3 10 70* 4 0 90* 5 10 70* 6 20 90* 7 0 50* 8 0 0average number 0.4   4.4* of days shedding*Within a time point, vaccinates different from non-vaccinates, P < 0.05by either Fisher's exact test (percent data) or Wilcoxon 2 sample test(days shedding).

The extent (severity and duration) of clinical signs of influenza amongvaccinates was substantially reduced relative to the controls. Thescores from clinical signs relevant to influenza and the objectivetemperature measurements both demonstrated a statistically significantreduction in the group of vaccinates when compared to those from thecontrol group; indicating that the vaccine conferred significantprotection from disease by the heterologous strain.

The ability of horses to shed influenza virus post-challenge was alsosignificantly reduced in vaccinates as opposed to controls in both theincidence of horses positive for shedding on certain days post-challengeand the mean number of days of shedding per horse. This decreasedshedding by vaccinates is important in that it should serve to reducethe potential for exposure of susceptible animals to the wild-type virusin an outbreak of influenza.

Overall, the results of this study show that the vaccine conferredprotection against a heterologous challenge by a member of the Eurasianlineage of equine influenza virus strains.

EXAMPLE 10

This Example discloses an animal study to evaluate the ability of atherapeutic composition comprising cold-adapted equine influenza virusEIV-P821 to aid in the prevention of disease following exposure to aheterologous strain of equine influenza virus.

The heterologous strain tested was A/equine/2/Kentucky/98[H3N8](obtained from Tom Chambers, University of Kentucky). Eight poniesaged 5 to 7 months were used for this efficacy study. The horses wereassigned to two groups, one group of 4 to be vaccinated and anothergroup of 4 to serve as non-vaccinated controls. Ponies were vaccinatedas described in Example 8, on Day 0.

Clinical observations were performed for the vaccinates on Study Day 0(before vaccination and at 4 hours post-vaccination), as well as on Days1 to 8, 23, 30 to 50, and 57 post-vaccination. Controls were observedclinically on Days 29 to 50 and 57. The observations were performed andscored as described in Example 8.

The challenge material i.e. equine flu strain from Kentucky/98, wasprepared by passing the isolated virus two times in eggs. The inoculumfor each horse was prepared by thawing 0.5 ml of the virus, thendiluting in 4.5 ml of sterile phosphate-buffered saline. The inoculumwas administered by nebulization using a mask for each individual horseon Day 36 post-vaccination.

The clinical observation scores were summed on each day for each horse,and horses were ranked according to the cumulative total score from days1 to 9 post-challenge. Theses results are shown in Table 24. TABLE 24Clinical sign observations: total scores, ranked by total score. HalterTotal Score^(#) Group Identity Days 1 to 9 post-challenge 1-Vaccinate 500 1-Vaccinate 52 0 1-Vaccinate 55 1 1-Vaccinate 15 2 2-Control 61 212-Control 20 25 2-Control 7 26 2-Control 13 26^(#)Total scores represent the sum of daily scores (where daily scoresequal the sum of scores for coughing, nasal discharge, respiration, anddepression) and are ranked from the lowest (least severe) to highest(most severe) scores.

The results of Table 24 show that the scores for vaccinates were between0 and 2, which was significantly lower than the score for controls,which were between 21 and 26.

Rectal temperatures were measured daily beginning 6 days prior tochallenge, and continuing until 9 days post-challenge. Day 0 is the dayrelative to challenge. Data from days 0 through 9 were included in theanalysis. These results are shown in Table 25. TABLE 25 Effect ofChallenge on daily mean temperatures (° C.) in vaccinated and controlhorses. Day post challenge control vaccinate difference 0 99.7 99.5 0.21 100.0 99.6 0.4 2 103.9 100.2 3.7 3 99.8 99.2 0.6 4 99.6 99.1 0.5 599.8 99.3 0.5 6 99.6 99.3 0.3 7 99.3 99.0 0.3 8 99.7 99.6 0.1 9 99.599.1 0.4

The temperatures of the control horses were higher than the temperaturesof the vaccinated horses on all days. The temperature in control horseswas significantly higher on day 2.

Nasopharyngeal swabs were collected on days 1 and 8, post-challenge, asdescribed in Example 3. These samples were tested for shed virus by anegg infectivity assay as described in Example 1. The results of theassay are shown in Table 26. TABLE 26 Virus shedding post-challengedetected by egg infectivity. Study day 35 37 38 39 40 41 42 43 44 Dayspost-challenge Identity −1 1 2 3 4 5 6 7 8 No. days positive Group No.Detection of virus* per horse Vaccinates 15 0 2 0 3 3 0 2 1 0 5 50 0 0 00 0 1 0 0 0 1 52 0 0 3 3 2 2 0 0 0 4 55 0 2 3 1 3 0 0 0 0 4 No. horsespositive per day 0 2 2 3 3 2 1 1 0 Controls 07 0 3 3 3 3 3 3 1 0 7 13 03 3 3 3 3 3 1 0 7 20 0 2 3 3 3 3 3 1 0 7 61 0 3 3 3 3 3 3 2 0 7 No.horses positive per day 0 4 4 4 4 4 4 4 0*Values refer to the number of eggs testing positive of 3 eggs testedper sample. For statistical analysis, a sample was considered positivefor virus if at least 1 egg was positive per sample.

The results of Table 26 show that the number of horses positive per daywas higher for the controls than for the vaccinates. Additionally,control horses were positive for more days than vaccinates.

The scores from clinical signs relevant to influenza and the objectivetemperature measurements both demonstrated significant differences inthe group of vaccinates when compared to the control group; this showsthat the vaccine conferred significant protection from disease caused bythe heterologous strain Kentucky/98.

The ability of horses to shed influenza virus post-challenge was alsosignificantly reduced in vaccinated as opposed to controls in the meannumber of days of shedding per horse. This decreased shedding byvaccinates is important in that it should serve to reduce the potentialfor exposure of susceptible animals to the wild-type virus in anoutbreak of influenza.

Overall, the results of this study show that the vaccine safelyconferred protection to a heterologous challenge by a recent andclinically relevant isolate. When the results of this study are viewedin the light of the protection previously demonstrated againstheterologous challenge with a Eurasian strain (Example 9), there isclear evidence to support the assertion that this modified live vaccinecan confer protection against heterologous as well as homologous equineinfluenza infection.

EXAMPLE 11

This example describes the cloning and sequencing of equine influenza M(matrix) protein nucleic acid molecules for wild type and cold-adaptedequine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus M protein, were produced as follows. A PCR productcontaining an equine M gene was produced by PCR amplification fromequine influenza virus DNA, and primers w584 and w585, designated SEQ IDNO:26, and SEQ ID NO:27, respectively. A nucleic acid molecule of 1023nucleotides, denoted nei_(wt)M₁₀₂₃, with a coding strand having anucleic acid sequence designated SEQ ID NO:1 was produced by further PCRamplification using the above described PCR product as a template andcloned into pCR 2.1®TA cloning vector, available from Invitrogen,Carlsbad, Calif., using standard procedures recommended by themanufacturer. The primers used were the T7 primer, designated by SEQ IDNO:29 and the REV primer, designated by SEQ ID NO:28. Plasmid DNA waspurified using a mini-prep method available from Qiagen, Valencia,Calif. PCR products were prepared for sequencing using a PRISM™ DyeTerminator Cycle Sequencing Ready Reaction kit, a PRISM™ drhodamineTerminator Cycle Sequencing Ready Reaction kit, or a PRISM™ BigDye™Terminator Cycle Sequencing Ready Reaction kit, all available from PEApplied Biosystems, Foster City, Calif., following the manufacturer'sprotocol. Specific PCR conditions used with the kit were a rapid ramp to95° C., hold for 10 seconds followed by a rapid ramp to 50° C. with a 5second hold then a rapid ramp to 60° C. with a 4 minute hold, repeatingfor 25 cycles. Different sets of primers were used in differentreactions: T7 and REV were used in one reaction; w584 and w585 were usedin a second reaction; and efM-a1, designated SEQ ID NO:31 and efM-s1,designated SEQ ID NO:30 were used in a third reaction. PCR products werepurified by ethanol/magnesium chloride precipitation. Automatedsequencing of DNA samples was performed using an ABI PRISM™ Model 377with XL upgrade DNA Sequencer, available from PE Applied Biosystems.

Translation of SEQ ID NO:1 indicates that nucleic acid moleculenei_(wt)M₁₀₂₃ encodes a full-length equine influenza M protein of about252 amino acids, referred to herein as Pei_(wt)M₂₅₂, having amino acidsequence SEQ ID NO:2, assuming an open reading frame in which theinitiation codon spans from nucleotide 25 through nucleotide 28 of SEQID NO:1 and the termination codon spans from nucleotide 781 throughnucleotide 783 of SEQ ID NO:1. The region encoding Pei_(wt)M₂₅₂,designated nei_(wt)M₇₅₆, and having a coding strand comprisingnucleotides 25 to 780 of SEQ ID NO:1, is represented by SEQ ID NO:3.

SEQ ID NO:1 and SEQ ID NO:3 represent the consensus sequence obtainedfrom two wild type nucleic acid molecules, which differ in onenucleotide. Nucleotide 663 of nei_(wt1)M₁₀₂₃, i.e., nucleotide 649 ofnei_(wt1)M₇₅₆, was adenine, while nucleotide 663 of nei_(wt2)M₁₀₂₃,i.e., nucleotide 649 of nei_(wt2)M₇₅₆, was guanine. Translation of thesesequences does not result in an amino acid change at the correspondingamino acid; both translate to valine at residue 221 in Pei_(wt)M₂₅₂,

B. A nucleic acid molecule of 1023 nucleotides encoding a cold-adaptedequine influenza virus M, denoted nei_(ca1)M₁₀₂₃, with a coding strandhaving a sequence designated SEQ ID NO:4 was produced by further PCRamplification and cloned into the pCR®-Blunt cloning vector availablefrom Invitrogen, using conditions recommended by the manufacturer, andprimers T7 and REV. Plasmid DNA purification and cycle sequencing wereperformed as described in Example 11, part A. Translation of SEQ ID NO:4indicates that nucleic acid molecule nei_(ca1)M₁₀₂₃ encodes afull-length equine influenza M protein of about 252 amino acids,referred to herein as Pei_(ca1)M₂₅₂, having amino acid sequence SEQ IDNO:5, assuming an open reading frame in which the initiation codon spansfrom nucleotide 25 through nucleotide 28 of SEQ ID NO:4 and thetermination codon spans from nucleotide 781 through nucleotide 783 ofSEQ ID NO:4. The region encoding Pei_(ca1)M₂₅₂, designatednei_(ca1)M₇₅₆, and having a coding strand comprising nucleotides 25 to780 of SEQ ID NO:4, is represented by SEQ ID NO:6. PCR amplification ofa second nucleic acid molecule encoding a cold-adapted equine influenzaM protein in the same manner resulted in molecules nei_(ca2)M₁₀₂₃,identical to nei_(ca1)M₁₀₂₃, and nei_(ca2)M₇₅₆, identical tonei_(ca1)M₇₅₆.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)M₁₀₂₃ (SEQ ID NO:1) and nei_(ca1)M₁₀₂₃ (SEQ ID NO:4) by DNAalignment reveals the following differences: a G to T shift at base 67,a C to T shift at base 527, and a C to C shift at base 886. Comparisonof the amino acid sequences of proteins Pei_(wt)M₂₅₂ (SEQ ID NO:2) andPei_(ca1)M₂₅₂ (SEQ ID NO:5) reveals the following differences: a V to Lshift at amino acid 23 relating to the G to T shift at base 67 in theDNA sequences; and a T to I shift at amino acid 187 relating to the C toT shift at base 527 in the DNA sequences.

EXAMPLE 12

This example describes the cloning and sequencing of equine influenza HA(hemagglutinin) protein nucleic acid molecules for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus HA proteins were produced as follows. A PCR productcontaining an equine HA gene was produced by PCR amplification fromequine influenza virus DNA and primers w578 and w579, designated SEQ IDNO:32 and SEQ ID NO:33, respectively. A nucleic acid molecule of 1762nucleotides encoding a wild-type HA protein, denoted nei_(wt)HA₁₇₆₂,with a coding strand having a nucleic acid sequence designated SEQ IDNO:7 was produced by further PCR amplification using the above-describedPCR product as a template and cloned into pCR 2.1®TA cloning vector asdescribed in Example 11A. Plasmid DNA was purified and sequenced as inExample 11A, except that primers used in the sequencing kits were eitherT7 and REV in one case, or HA-1, designated SEQ ID NO:34, and HA-2,designated SEQ ID NO:35, in a second case.

Translation of SEQ ID NO:7 indicates that nucleic acid moleculenei_(wt)HA₁₇₆₂ encodes a full-length equine influenza HA protein ofabout 565 amino acids, referred to herein as Pei_(wt)HA₅₆₅, having aminoacid sequence SEQ ID NO:8, assuming an open reading frame in which theinitiation codon spans from nucleotide 30 through nucleotide 33 of SEQID NO:7 and the termination codon spans from nucleotide 1725 throughnucleotide 1727 of SEQ ID NO:7. The region encoding Pei_(wt)HA₅₆₅,designated nei_(wt)HA₁₆₉₅, and having a coding strand comprisingnucleotides 30 to 1724 of SEQ ID NO:7 is represented by SEQ ID NO:9.

B. A nucleic acid molecule of 1762 nucleotides encoding a cold-adaptedequine influenza virus HA protein, denoted nei_(ca1)HA₁₇₆₂, with acoding strand having a sequence designated SEQ ID NO:10 was produced asdescribed in Example 1113. Plasmid DNA purification and cycle sequencingwere performed as described in Example 12, part A.

Translation of SEQ ID NO:10 indicates that nucleic acid moleculenei_(ca1)HA₁₇₆₂ encodes a full-length equine influenza HA protein ofabout 565 amino acids, referred to herein as Pei_(ca1)HA₅₆₅, havingamino acid sequence SEQ ID NO:11, assuming an open reading frame inwhich the initiation codon spans from nucleotide 30 through nucleotide33 of SEQ ID NO:10 and the termination codon spans from nucleotide 1725through nucleotide 1727 of SEQ ID NO:10. The region encodingPei_(ca1)HA₅₆₅, designated nei_(ca1)HA₁₆₉₅, and having a coding strandcomprising nucleotides 30 to 1724 of SEQ ID NO:10, is represented by SEQID NO:12.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza HA protein in the same manner resulted inmolecules nei_(ca2)HA₁₇₆₂, identical to nei_(ca1)HA₁₇₆₂, andneica₂HA₁₆₉₅, identical to nei_(ca1)HA₁₆₉₅.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)HA₁₇₆₂ (SEQ ID NO:7) and nei_(ca1)HA₁₇₆₂ (SEQ ID NO:10) by DNAalignment reveals the following differences: a C to T shift at base 55,a G to A shift at base 499, a G to A shift at base 671, a C to T shiftat base 738, a T to C shift at base 805, a G to A shift at base 1289,and an A to G shift at base 1368. Comparison of the amino acid sequencesof proteins Pei_(ca1)HA₅₆₅ (SEQ ID NO:8) and Pei_(ca1)HA₅₆₅ (SEQ IDNO:11) reveals the following differences: a P to L shift at amino acid18 relating to the C to T shift at base 55 in the DNA sequences; a G toE shift at amino acid 166 relating to the & to A shift at base 499 inthe DNA sequences; an R to W shift at amino acid 246 relating to the Cto T shift at base 738 in the DNA sequences; an M to T shift at aminoacid 268 relating to the T to C shift at base 805 in the DNA sequences;a K to E shift at amino acid 456 relating to the A to G shift at base1368 in the DNA sequences. There is no change of the serine (S) atresidue 223 relating to the G to A shift at base 671 in the DNAsequences, nor is there a change of the arginine (R) at residue 429relating to the G to A shift at base 1289 in the DNA sequences.

EXAMPLE 13

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid moleculescorresponding to the N-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2-N proteins were produced as follows. A PCR productcontaining an N-terminal portion of the equine PB2 gene was produced byPCR amplification from equine influenza virus DNA, and primers w570 andw571, designated SEQ ID NO:36 and SEQ ID NO:37, respectively. A nucleicacid molecule of 1241 nucleotides encoding a wild type PB2-N protein,denoted nei_(et)PB2-N₁₂₄₁, with a coding strand having a nucleic acidsequence designated SEQ ID NO:13 was produced by further PCRamplification using the above described PCR product as a template andcloned as described in Example 11B. Plasmid DNA was purified andsequenced as in Example 11B, except that only T7 and REV primers wereused in the sequencing kits.

Translation of SEQ ID NO:13 indicates that nucleic acid moleculenei_(wt)PB2-N₁₂₄₁ encodes an N-terminal portion of influenza PB2 proteinof about 404 amino acids, referred to herein as P_(wt)PB2-N₄₀₄, havingamino acid sequence SEQ ID NO:14, assuming an open reading frame inwhich the initiation codon spans from nucleotide 28 through nucleotide30 of SEQ ID NO:13, and the last codon spans from nucleotide 1237through nucleotide 1239. The region encoding P_(wt)PB2-N₄₀₄, designatednei_(wt)PB2-N₁₂₁₄, and having a coding strand comprising nucleotides 28to 1239 of SEQ ID NO:13 is represented by SEQ ID NO:15.

B. A nucleic acid molecule of 1239 nucleotides encoding an N-terminalportion of influenza PB2 cold-adapted equine influenza virus PB2-Nprotein, denoted nei_(ca1)PB2-N₁₂₄₁, with a coding strand having asequence designated SEQ ID NO:16 was produced, and sequenced asdescribed in as in Example 12, part A.

Translation of SEQ ID NO:16 indicates that nucleic acid moleculenei_(ca1)PB2-N₁₂₄₁ encodes an N-terminal portion of equine influenzaPB-2 protein of about 404 amino acids, referred to herein asP_(ca1)PB2-N₄₀₄, having amino acid sequence SEQ ID NO:17, assuming anopen reading frame in which the initiation codon spans from nucleotide28 through nucleotide 30 of SEQ ID NO:16, and the last codon spans fromnucleotide 1237 through nucleotide 1239. The region encodingP_(ca1)PB2-N₄₀₄, designated nei_(ca1)PB2-N₁₂₁₄, and having a codingstrand comprising nucleotides 28 to 1239 of SEQ ID NO:16, is representedby SEQ ID NO:18.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB2-N protein in the same manner resultedin molecules nei_(ca2)PB2-N₁₂₄₁, identical to nei_(ca1)PB2-N₁₂₄₁, andnei_(ca2)PB2-N₁₂₁₄, identical to nei_(ca1)PB2-N₁₂₁₄.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB2-N₁₂₄₁ (SEQ ID NO:13) and nei_(ca1)PB2-N₁₂₄₁ (SEQ ID NO:16)by DNA alignment reveals the following difference: a T to C base shiftat base 370. Comparison of the amino acid sequences of proteinsP_(wt)PB2-N₄₀₄ (SEQ ID NO:14) and P_(ca1)PB2-N₄₀₄ (SEQ ID NO:17) revealsthe following difference: a Y to H shift at amino acid 124 relating tothe a T to C shift at base 370 in the DNA sequence.

EXAMPLE 14

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid moleculescorresponding to the C-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2-C proteins were produced as follows. A PCR productcontaining the C-terminal portion of the equine PB2 gene was produced byPCR amplification using from equine influenza virus DNA and primers w572and w573, designated SEQ ID NO:38 and SEQ ID NO:39, respectively. Anucleic acid molecule of 1233 nucleotides encoding a wild type PB2-Cprotein, denoted nei_(wt)PB2-C₁₂₃₃, with a coding strand having anucleic acid sequence designated SEQ ID NO: 19 was produced by furtherPCR amplification using the above-described PCR product as a templateand cloned as described in Example 11B. Plasmid DNA was purified andsequenced as in Example 11A, except that different primers were used inthe sequencing kits. T7 and REV were used in one instance; efPB2-a1,designated SEQ ID NO:40 and efPB2-s1, designated SEQ ID NO:41 were usedin another instance, and efPB2-a2, designated SEQ ID NO:42 and efPB2-s2,designated SEQ ID NO:43 were used in another instance.

Translation of SEQ ID NO:19 indicates that nucleic acid moleculenei_(wt1)PB2-C₁₂₃₃ encodes a C-terminal portion of influenza PB2 proteinof about 398 amino acids, referred to herein as P_(wt)PB2-C₃₉₈, havingamino acid sequence SEQ ID NO:20, assuming an open reading frame havinga first codon spans from nucleotide 3 through nucleotide 5 and atermination codon which spans from nucleotide 1197 through nucleotide1199 of SEQ ID NO:19. Because SEQ ID NO:19 is only a partial genesequence, it does not contain an initiation codon. The region encodingP_(wt)PB2-C₃₉₈, designated nei_(wt)PB2-C₁₁₉₄, and having a coding strandcomprising nucleotides 3 to 1196 of SEQ ID NO:19 is represented by SEQID NO:21.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB2-N protein in the same manner resulted in a nucleicacid molecule of 1232 nucleotides denoted nei_(wt2)PB2-N₁₂₃₂, with acoding strand with a sequence designated SEQ ID NO:22.nei_(wt2)PB2-N₁₂₃₂ is identical to nei_(wt1)PB2-C₁₂₃₃, expect thatnei_(wt2)PB2-N₁₂₃₂ lacks one nucleotide on the 5′-end. Translation ofSEQ ID NO:22 indicates that nucleic acid molecule nei_(wt1)PB2-C₁₂₃₃also encodes P_(wt)PB2-C₃₉₈(SEQ ID NO:20), assuming an open readingframe having a first codon which spans from nucleotide 2 throughnucleotide 4 and a termination codon spans from nucleotide 1196 throughnucleotide 1198 of SEQ ID NO:22. Because SEQ ID NO:22 is only a partialgene sequence, it does not contain an initiation codon. The nucleic acidmolecule having a coding strand comprising nucleotides 2 to 1195 of SEQID NO:22, denoted nei_(wt2)PB2-C₁₁₉₄, is identical to SEQ ID NO:21.

B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminalportion of influenza PB2 cold-adapted equine influenza virus protein,denoted nei_(ca1)PB2-C₁₂₃₂, and having a coding strand having a sequencedesignated SEQ ID NO:23 was produced as described in as in Example 14,part A, except that the pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:23 indicates that nucleic acid moleculenei_(ca1)PB2-C₁₂₃₂ encodes a C-terminal portion of equine influenza PB-2protein of about 398 amino acids, referred to herein as P_(ca1)PB2-C₃₉₈,having amino acid sequence SEQ ID NO:24, assuming an open reading framehaving a first codon which spans from nucleotide 2 through nucleotide 4and a termination codon spans from nucleotide 1196 through nucleotide1198 of SEQ ID NO:23. Because SEQ ID NO:23 is only a partial genesequence, it does not contain an initiation codon. The region encodingP_(ca1)PB2-C₃₉₈, designated nei_(ca1)PB2-C1194, and having a codingstrand comprising nucleotides 2 to 1195 of SEQ ID NO:23, is representedby SEQ ID NO:25.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB2-C protein in the same manner resultedin molecules nei_(ca2)PB2-C₁₂₃₁, containing one less nucleotide at the3′ end than nei_(ca1)PB2-N₁₂₄₁; and nei_(ca2)PB2-N₁₂₁₄, identical tonei_(ca1)PB2-N₁₂₁₄,

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) and nei_(wt1) PB2-C₁₂₃₂ (SEQ ID NO:23)by DNA alignment reveals the following differences: an A to C base shiftat base 153 of SEQ ID NO:19, and a G to A base shift at base 929 of SEQID NO:19. Comparison of the amino acid sequences of proteinsP_(wt)PB2-C₃₉₈ (SEQ ID NO:20) and P_(ca1)PB2-₃₉₈ (SEQ ID NO:24) revealsthe following difference: a K to Q shift at amino acid 51 when relatingto the an A to C base shift at base 153 in the DNA sequences. There isno amino acid shift resulting from the G to A base shift at base 929.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1-38. (canceled)
 39. An isolated equine influenza protein, wherein saidequine influenza protein comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:14, SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:24.
 40. The protein ofclaim 39, wherein said protein is encoded by a nucleic acid moleculeselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:25.
 41. A therapeuticcomposition to protect an animal against disease caused by an influenzaA virus, said composition comprising an isolated equine influenzaprotein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQID NO:17, SEQ ID NO:20, and SEQ ID NO:24.
 42. The composition of claim41, wherein said protein is encoded by a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, and SEQ ID NO:25.
 43. A method to protect an animalagainst disease caused by an influenza A virus comprising administeringto said animal a therapeutic composition comprising an isolated equineinfluenza protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14,SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:24.
 44. The method of claim43, wherein said protein is encoded by a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO: 23, and SEQ ID NO:25.